X chromosome specific molecular marker of micropterus salmoides and application of X chromosome specific molecular marker

Abstract

The invention discloses an X-chromosome specific molecular marker of micropterus salmoides and application of the X-chromosome specific molecular marker. On the basis of a whole genome sequencing technology, the X chromosome specific molecular marker of micropterus salmoides is obtained. Aiming at the molecular marker, the invention also provides a detection primer pair and a detection method of the molecular marker, and by detecting the molecular marker, rapid and accurate screening of YY supermale fish individuals can be realized to obtain a YY supermale fish population. The obtained YY super-male fish group and a proper female fish group are jointly fed and bred, and then the micropterus salmoides all-male fingerling can be obtained. If the YY super-male fish group and the YY pseudo-female fish group are jointly bred and bred, largemouth bass offspring which is all YY super-male fish can be obtained. Compared with a traditional all-male breeding method of the largemouth black bass, the all-male breeding method of the largemouth black bass is beneficial to simplifying all-male breeding steps of the largemouth black bass, shortening breeding time, and beneficial to cultivation of all-male largemouth black bass fingerlings and popularization of single-male breeding of the largemouth black bass.

Description

Technical Field

[0001] This invention belongs to the field of fish sex identification and fish fry and fingerling breeding technology. More specifically, it relates to an X chromosome-specific molecular marker for largemouth bass and its application. Background Technology

[0002] Largemouth bass ( Micropterus salmoides The largemouth bass, commonly known as California bass, is highly adaptable, fast-growing, and easy to catch. Its delicious, tender flesh, free of intramuscular bones, makes it popular with farmers and consumers, making it an important freshwater aquaculture species. However, largemouth bass exhibits sexual dimorphism in growth, heat tolerance, and disease resistance; males grow faster than females, with one-year-old males weighing 1.2 times more than females. Furthermore, males show stronger resistance to frog iridovirus (LMBV) under high-temperature conditions. Additionally, largemouth bass reach sexual maturity in one year. In conventional largemouth bass farming, sexual behavior easily occurs when fish reach marketable size, leading to reproductive nutrient loss, postpartum syndrome, and increased mortality. Monosex farming can delay sexual maturity and reduce mortality due to postpartum syndrome. Therefore, breeding all-male largemouth bass and promoting monosex farming will yield better economic benefits.

[0003] Sex determination in fish is complex and diverse, influenced by both genetic factors (such as chromosomes) and environmental factors, with significant differences in sex determination mechanisms among different species. Sex determination in fish where sex is directly controlled by genes on chromosomes includes the XY type (male heterogamous), ZW type (female heterogamous), and polychromatic type. The largemouth bass exhibits the XY type of sex determination. The traditional method for breeding all-male XY-type fish is as follows: ① Sex reversal is achieved through estrogen-induced experiments (including XY and XX), followed by testcrossing with XY (♂) male fish (this process requires one-to-one pairing, which is currently difficult to achieve in largemouth bass) to select XY (♀) physiological females; ② The selected XY (♀) physiological females are testcrossed with XY (♂) male fish, and the offspring (including XY, XX, and YY) are then testcrossed with XX (♀) female fish to select YY (♂) supermales; ③ The selected YY (♂) supermales are paired with XX (♀) female fish to produce all-male XY (♂) fish in batches. This method of determining parental sex genotypes through testcrosses requires observation of sex glands in the offspring fry to confirm the parental sex genotypes, which is time-consuming, labor-intensive, and has a long breeding cycle.

[0004] Obtaining sex-linked molecular markers through genome-wide screening allows for efficient and precise identification of sex genotypes, significantly improving the detection efficiency of YY supermale fish and effectively saving manpower, resources, and shortening breeding time. While several sex-related molecular markers for largemouth bass have been reported, most can only distinguish between XX and XY species, lacking practical application in YY fish, or, based on their principles, are incapable of distinguishing between XY and YY, hindering rapid screening and identification of YY fish. Specifically, the reported sex-related molecular markers for largemouth bass can accurately distinguish between XY pseudo-females and XX females in the first generation of all-male largemouth bass breeding, allowing for the screening of XY pseudo-females. However, in the second generation of all-male largemouth bass breeding, there are XY males, XX females, and YY supermales in a 1:2:1 ratio; the reported sex-related molecular markers cannot effectively distinguish between XY and YY species in this case. Specific molecular markers on the X chromosome of largemouth bass can distinguish between XY and YY chromosomes, but these molecular markers are not easy to obtain.

[0005] Unlike birds and mammals, the sex chromosomes of many fish are less differentiated (Bachtrog D, Mank J E, Peichel C L, et al. Sex determination: why so many ways of doing it? [J]. PLoS biology, 2014, 12(7): e1001899.; Graves J A M. Evolution of vertebrate sex chromosomes and dosage compensation [J]. Nature Reviews Genetics, 2016, 17(1): 33-46.). In many fish species, the sex chromosome pair is the earliest originated homomorphic chromosome. For example, in XY male heterogametic fish, most of the Y chromosome is a pseudoautosomal region (PAR), which can recombine with the X chromosome, while the sex-determining region (SDR) only accounts for a small part of the sex chromosome. In addition, most fish have experienced a teleost genome duplication (TGD) event, which has greatly increased the complexity of their genomes and has a great impact on the accurate assembly of sex chromosomes. That is, through the conventional whole-genome sequencing method, whole-genome sequencing, assembly and alignment of female and male fish, it is not necessarily accurate to locate and assemble the complete X and Y sex chromosomes, and to screen the differential regions and fragments specific to X and Y, respectively. In addition, in the study of sex markers in many fish, it is found that it is more difficult to screen X chromosome markers (Liu S, Han C, Huang J, et al. Screening and characterization of X chromosome-specific markers in mandarin fish (Siniperca chuatsi) [J]. Aquaculture, 2023, 562: 738833.), and the verification of YY super-male fish test fish materials is required, while the acquisition of YY fish is also very difficult.

[0006] A molecular marker for identifying the genetic sex of Micropterus salmoides is disclosed in Chinese Patent Application No. CN114717301A. The molecular marker exists on the X chromosome of Micropterus salmoides and is absent on the Y chromosome. However, the size difference of the marker molecules of this XY chromosome mixed molecular marker is too small, only 66 bp, 14 bp and 34 bp, making it difficult to distinguish by electrophoresis detection. The detection results of XX and YY samples may both be only one band and the sizes are very close (345 bp and 314 bp). The effectiveness and accuracy of this marker have not been verified on YY super male fish test samples. Since the current main breeding method of Micropterus salmoides is group natural mating reproduction, if the detection accuracy of YY super male fish is not enough, there may be undetected XY male fish in the breeding pond, which will seriously affect the male rate of the offspring of the breeding stock, and it is difficult to remedy. Therefore, the X chromosome specific molecular marker of Micropterus salmoides is very important in the all-male breeding technology of Micropterus salmoides, and an X chromosome specific molecular marker capable of effectively distinguishing YY super male fish needs to be developed. SUMMARY

[0007] The present application provides an X chromosome specific molecular marker of Micropterus salmoides, which can accurately identify YY super male fish and promote the cultivation of all-male fish of Micropterus salmoides.

[0008] The first object of the present application is to provide an X chromosome specific molecular marker of Micropterus salmoides.

[0009] The second object of the present application is to provide a detection reagent for the molecular marker.

[0010] The third object of the present application is to provide the use of the molecular marker or the detection reagent in the detection of YY super male Micropterus salmoides.

[0011] The fourth object of the present application is to provide the use of the molecular marker or the detection reagent in the preparation of a product for detecting YY super male Micropterus salmoides.

[0012] The fifth object of the present application is to provide the use of the molecular marker or the detection reagent in the all-male breeding of Micropterus salmoides.

[0013] The sixth object of the present application is to provide a method for detecting YY super male Micropterus salmoides.

[0014] The seventh object of the present application is to provide an all-male breeding method of Micropterus salmoides.

[0015] The above objects of the present application are achieved by the following technical solutions: The inventor of the present application obtains a X chromosome specific molecular marker of Micropterus salmoides through sequencing and alignment analysis. The present application also provides a detection primer pair and a detection method for the molecular marker. The YY super male fish individuals can be quickly screened by detecting the molecular marker using the primer pair and the method, and the YY super male fish population is obtained, which promotes the all-male breeding of Micropterus salmoides. Therefore, the present application claims the molecular marker and its application.

[0016] The present application provides a X chromosome specific molecular marker of Micropterus salmoides, which is located on the X chromosome of Micropterus salmoides and comprises a DNA fragment 1 and a DNA fragment 2. The reference sequence of the X chromosome of Micropterus salmoides in the NCBI database is NW_024040041.1. The DNA fragment 1 is located at the 34801783-348001930 nucleotides of the X chromosome of Micropterus salmoides NW_024040041.1, and the nucleotide sequence thereof is shown in SEQ ID NO. 2. The DNA fragment 2 is located at the 34803159-34803261 nucleotides of the X chromosome of Micropterus salmoides NW_024040041.1, and the nucleotide sequence thereof is shown in SEQ ID NO. 3.

[0017] The present application also provides a detection reagent of the molecular marker.

[0018] Specifically, the detection reagent is a detection kit of the molecular marker, and the kit contains a primer pair for amplifying the molecular marker.

[0019] Specifically, the primer pair comprises a primer pair for amplifying the DNA fragment 1 and the DNA fragment 2 which are designed based on the sequences on both sides of the DNA fragment 1 and the DNA fragment 2, respectively, and a primer pair obtained by designing upstream primers and downstream primers with the DNA fragment 1 and the DNA fragment 2 as targets, respectively.

[0020] In a specific embodiment of the present application, the nucleotide sequences of the primer pair are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively, which are obtained by designing upstream primers and downstream primers with the DNA fragment 1 and the DNA fragment 2 as targets, respectively.

[0021] Since the molecular marker of the present application only exists on the X chromosome of Micropterus salmoides and does not exist on the Y chromosome of Micropterus salmoides, it is a X chromosome specific molecular marker of Micropterus salmoides, and YY super male Micropterus salmoides can be accurately identified using the same. Therefore, the present application claims the application of the molecular marker or the detection reagent in detecting YY super male Micropterus salmoides.

[0022] The present application also claims the application of the molecular marker or the detection reagent in preparing a product for detecting YY super male Micropterus salmoides.

[0023] The application also claims the use of the molecular marker or the detection reagent in the all-male breeding of Micropterus salmoides.

[0024] The application also provides a method for detecting YY super-male Micropterus salmoides, which comprises the following steps: S1. Collecting the tissue sample of the fish to be tested and extracting the genomic DNA thereof; S2. Using the obtained genomic DNA as a template, performing PCR amplification by using the primer pair for amplifying the molecular marker; S3. Performing electrophoresis detection on the PCR amplification product obtained in S2, and the absence of an amplification band indicates YY super-male Micropterus salmoides.

[0025] Alternatively, the nucleotide sequences of the primer pair are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0026] The application also provides an all-male breeding method of Micropterus salmoides, which comprises the following steps:

[0027] Specifically, the all-male breeding method comprises the following steps: S1. Co-bred XX female fish (♀) and XY male fish (♂) of Micropterus salmoides, and performing estrogen treatment sex reversal on the offspring to obtain offspring half of which are XY pseudo-female fish (♀) and half of which are XX female fish (♀) of Micropterus salmoides, and screening XY pseudo-female fish (♀) individuals by using the Y chromosome sex molecular marker of Micropterus salmoides; S2. Co-bred XY pseudo-female fish (♀) and XY male fish (♂) of Micropterus salmoides, and obtain offspring with a ratio of 1:2:1 of YY super-male fish (♂), XY male fish (♂) and XX female fish (♀), and screening YY super-male fish (♂) individuals by using the X chromosome specific molecular marker of Micropterus salmoides; S3. Co-bred XY pseudo-female fish (♀) and YY super-male fish (♂) of Micropterus salmoides, and breed offspring half of which are subjected to estrogen treatment sex reversal to obtain offspring half of which are XY pseudo-female fish (♀) and half of which are YY pseudo-female fish (♀) of Micropterus salmoides, and screening YY pseudo-female fish (♀) individuals by using the X chromosome specific molecular marker of Micropterus salmoides; and screening YY super-male fish (♂) individuals by using the X chromosome specific molecular marker of Micropterus salmoides; S4. YY pseudo-female (♀) and YY super-male (♂) are co-bred, and all the offspring are YY super-male (♂) individuals, so that YY super-male (♂) species can be mass-produced; or XX female (♀) and YY super-male (♂) are co-bred, and all the offspring are XY male (♂) individuals, so that all-male Micropterus salmoides fry can be mass-produced.

[0028] The present application has the following beneficial effects: Based on the whole genome sequencing technology, the present application obtains an X chromosome specific molecular marker of Micropterus salmoides. For the molecular marker, the present application further provides a detection primer pair and a detection method. The rapid and accurate screening of YY super-male individuals can be realized by detecting the molecular marker, and a YY super-male population is obtained. YY super-male species can be obtained by co-bred YY super-male population and suitable female (pseudo-female) population. If YY super-male population and YY pseudo-female population are co-bred, Micropterus salmoides offspring all of which are YY super-male can be obtained. Compared with the traditional all-male breeding method of Micropterus salmoides, the present application is helpful to simplify the all-male breeding steps of Micropterus salmoides and shorten the breeding time. Without the molecular marker, each test fish to be identified needs to be paired with an ordinary XX female one by one in the all-male breeding process, and each group of paired offspring is bred separately. After the gonadal development of the offspring can be distinguished by naked eyes, the genetic sex of the test fish to be identified is determined by the offspring sex feedback. This process is complicated and time-consuming. The artificial pairing and breeding of Micropterus salmoides is difficult. The use of the molecular marker of the present application avoids the tedious test cross breeding program of artificial pairing and one-to-one breeding, and can also be screened early, truly saving time and effort.

[0029] The present application is helpful to the cultivation of all-male Micropterus salmoides and the popularization of Micropterus salmoides monosex culture. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 4 is a detection result of using primer pair LMBX-F and LMBX-R to detect Micropterus salmoides YY super-male, XX female and XY male. In the figure, M is a DNA molecular weight standard StarMarker D2000.

[0031] Figure 2 Figure 6 is a technical roadmap of the all-male Micropterus salmoides breeding method. DETAILED DESCRIPTION

[0032] The present application will be further described below in combination with the drawings and specific examples in the specification, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] Example 1 Obtaining of X chromosome specific molecular markers of Micropterus salmoides The process for obtaining the X chromosome specific molecular markers of Micropterus salmoides according to the present application is as follows: S1. Sequencing (PacBio single molecule real-time sequencing and Hi-C technology) of high-quality chromosome-level Micropterus salmoides female (XX, GenBank assembly GCA_014851395.1) and male (XY, GenBank assembly GCA_036785525.1) whole genomes was performed to obtain complete X chromosome and Y chromosome sequence structures.

[0035] S2. Thirty female (XX) and male (XY) Micropterus salmoides were selected from multiple local populations, and genomic DNA was extracted to construct a 350-500 bp fragment sequencing library. Pair-End PE150 sequencing was performed using the DNBSEQ-T7 platform to obtain Micropterus salmoides female (XX) and male (XY) genomic sequencing clean data. The library construction steps are described in detail in the BGI NGSDNA Library Prep Kit instructions, and sequencing was performed by Shenzhen Huada Gene Technology Co., Ltd.

[0036] S3. The Micropterus salmoides female and male reference genomes and female and male resequencing reads were aligned using bwa-0.7.17.tar.bz2 (https: / / bio-bwa.sourceforge.net / ) software, using a double-end alignment strategy and default parameters. Sequencing depth analysis and screening were performed using SAMtools v1.4 software, and X chromosome specific candidate DNA fragments were selected. PCR primers were designed based on the selected X chromosome specific candidate DNA fragments, and the primers were synthesized by Shengong Bioengineering Co., Ltd.

[0037] S4. Four caudal fins each from female (XX), male (XY), and super-male (YY) largemouth bass whose sex was confirmed by dissection were harvested. Genomic DNA samples were prepared according to the instructions, and their quality and concentration were determined by 1% agarose gel electrophoresis and a TECAN infinite200 microplate reader. Using the genomic DNA from these 12 individuals as templates, PCR detection was performed using the PCR primers designed and synthesized in S3. The PCR amplification reaction system was: 10 μL of 2×Taq PCR StarMix (Dye), 0.4 μL each of 10 μmol / L forward and reverse primers, 1 μL of template DNA (100 ng DNA), and 8.2 μL of ddH2O, for a total volume of 20 μL. After mixing, the mixture was centrifuged. The PCR amplification reaction program was: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, and 72℃ extension for 80 s, for a total of 37 cycles; and a final extension at 72℃ for 10 min. min; the validity of the X chromosome-specific candidate DNA fragment was determined by agarose gel electrophoresis of the PCR amplification product; when the PCR amplification and electrophoresis results showed that both female (XX) and male (XY) individuals had specific amplification bands of the X chromosome, while no specific amplification bands of the X chromosome were detected in super males (YY), then the fragment was determined to be an X chromosome-specific molecular marker of the largemouth bass.

[0038] Through a series of experiments, this invention obtained an X chromosome-specific molecular marker for largemouth bass. The molecular marker is located on the X chromosome of the largemouth bass, and the reference sequence of the largemouth bass X chromosome in the NCBI database is NW_024040041.1. The nucleotide sequence of the DNA fragment containing the molecular marker is shown in SEQ ID NO.1. Specifically, the molecular marker of this invention includes DNA fragment 1 and DNA fragment 2; DNA fragment 1 is located at nucleotides 34801783–348001930 of the largemouth bass X chromosome NW_024040041.1, and its nucleotide sequence is shown in SEQ ID NO.2; DNA fragment 2 is located at nucleotides 34803159–34803261 of the largemouth bass X chromosome NW_024040041.1, and its nucleotide sequence is shown in SEQ ID NO.3.

[0039] Example 2: A method for detecting male largemouth bass (YY type). Based on the molecular marker described in Example 1, this invention designed an upstream primer F (LMBX-F) targeting DNA fragment 1 and a downstream primer R (LMBX-R) targeting DNA fragment 2, obtaining a primer pair capable of detecting the molecular marker and thus detecting male largemouth bass YY. The nucleotide sequences of the primer pair are shown below: LMBX-F: 5'-GGGACATTTTTGATCATTCAATCCAT-3' (SEQ ID NO. 4); LMBX-R: 5'-TGAGATGCTGCTGCTCTAGT-3' (SEQ ID NO.5); Based on the primer pair, this invention provides a method for detecting the molecular marker, or a method for detecting YY male largemouth bass, comprising the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Using the obtained genomic DNA as a template, perform PCR amplification using the primer pairs LMBX-F and LMBX-R; S3. Electrophoresis was performed on the PCR amplification products. The fish with no bands were identified as male largemouth bass (YY).

[0040] The PCR amplification reaction system consisted of: 10 μL of 2×Taq PCR StarMix (Dye), 0.4 μL each of 10 μmol / L upstream and downstream primers, 1 μL of template DNA (100 ng DNA), and 8.2 μL of ddH2O, for a total volume of 20 μL. The PCR amplification program was as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, and 72℃ extension for 80 s, for a total of 37 cycles; and a final extension at 72℃ for 10 min.

[0041] To test the accuracy of the method for detecting YY male largemouth bass, this invention randomly selected 8 normal and healthy YY super-male largemouth bass, 4 XX female largemouth bass, and 4 XY male largemouth bass. Fin samples were collected from each sample, and genomic DNA was extracted. PCR amplification was performed using the aforementioned primer pairs and method, and the amplified products were detected by electrophoresis. The detection results of YY super-male, XX female, and XY male largemouth bass using primer pairs LMBX-F and LMBX-R are as follows: Figure 1 As shown. By Figure 1 It can be seen that the fin samples of YY super-male largemouth bass did not have specific amplification bands, while the fin samples of XX females and XY males all had specific amplification bands. This indicates that the primer pairs and methods described in this invention can accurately detect and screen YY super-male largemouth bass.

[0042] Example 3: A method for breeding all-male largemouth bass Based on the molecular markers and their detection methods described in this invention, this invention also provides a method for breeding all-male largemouth bass, the technical roadmap of which is shown below. Figure 2 As shown.

[0043] Combination Figure 2It can be known that the large mouth bass all-male breeding method comprises the following steps: S1. XX female fish (♀) and XY male fish (♂) of the large mouth bass are jointly bred, and the offspring seedlings are subjected to estrogen treatment sex reversal to obtain offspring half of which are XY pseudo-female fish (♀) and half of which are XX female fish (♀) of the large mouth bass, and XY pseudo-female fish (♀) individuals are screened out by using Y chromosome sex molecular markers of the large mouth bass; S2. XY pseudo-female fish (♀) and XY male fish (♂) of the large mouth bass are jointly bred to obtain YY super-male fish (♂), XY male fish (♂) and XX female fish (♀) offspring in a ratio of 1:2:1, and YY super-male fish (♂) individuals are screened out by using the X chromosome specific molecular markers of the large mouth bass; S3. XY pseudo-female fish (♀) and YY super-male fish (♂) of the large mouth bass are jointly bred, and half of the offspring are subjected to estrogen treatment sex reversal to obtain offspring half of which are XY pseudo-female fish (♀) and half of which are YY pseudo-female fish (♀) of the large mouth bass, and YY pseudo-female fish (♀) individuals are screened out by using the X chromosome specific molecular markers of the large mouth bass; and the other half is screened out by using the X chromosome specific molecular markers of the large mouth bass; S4. YY pseudo-female fish (♀) and YY super-male fish (♂) of the large mouth bass are jointly bred to obtain YY super-male fish (♂) individuals, and batch production of YY super-male fish (♂) can be carried out; or XX female fish (♀) and YY super-male fish (♂) of the large mouth bass are jointly bred to obtain XY male fish (♂) individuals, and batch production of large mouth bass all-male seedlings can be carried out.

[0044] Compared with the traditional large mouth bass all-male breeding method, the YY (♂) super-male fish can be quickly obtained by cutting fin bar samples of the offspring to be detected for ordinary PCR amplification, without the need of crossing screened XY (♀) physiological female fish with XY (♂) ordinary male fish, and then crossing the obtained offspring (including XY, XX and YY) with XX (♀) ordinary female fish to screen out YY (♂) super-male fish, which greatly simplifies the breeding steps.

[0045] The above embodiment is a preferred embodiment of the present application, but the embodiment of the present application is not limited by the above embodiment, and any change, modification, substitution, combination and simplification made without departing from the spirit and principle of the present application shall be equivalent replacement mode and shall be included in the protection scope of the present application.

Claims

1. A microsatellite marker specific for the X chromosome of a largemouth bass, characterized in that, The molecular marker is located on the X chromosome of the largemouth bass, and includes DNA segment 1 and DNA segment 2; the reference sequence of the X chromosome of the largemouth bass in the NCBI database is NW_024040041.1; the segment 1 is located at the nucleotides 34801783-348001930 of the X chromosome of the largemouth bass NW_024040041.1, and the nucleotide sequence is shown as SEQ ID NO. 2; the segment 2 is located at the nucleotides 34803159-34803261 of the X chromosome of the largemouth bass NW_024040041.1, and the nucleotide sequence is shown as SEQ ID NO.

3.

2. The detection reagent of the molecular marker of claim 1.

3. The detection reagent according to claim 2, characterized in that, The detection reagent is a detection kit of the molecular marker, which contains a primer pair for amplifying the molecular marker.

4. The test reagent according to claim 3, characterized in that, The nucleotide sequences of the primer pair are shown as SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

5. The application of the molecular marker of claim 1 or the detection reagent of any one of claims 2-4 in detecting YY super-male largemouth bass.

6. The application of the molecular marker of claim 1 or the detection reagent of any one of claims 2-4 in preparing a product for detecting YY super-male largemouth bass.

7. The application of the molecular marker of claim 1 or the detection reagent of any one of claims 2-4 in the all-male breeding of largemouth bass.

8. A method of detecting a YY super-male largemouth bass, characterized by, The method comprises the following steps: S1. Collecting tissue samples of the fish to be tested and extracting genomic DNA therefrom; S2. Using the obtained genomic DNA as a template, and performing PCR amplification by using a primer pair for amplifying the molecular marker of claim 1; S3. Performing electrophoresis detection on the PCR amplification product obtained in S2, and the fish without amplification band is a YY super-male largemouth bass.

9. The method of claim 8, wherein, The nucleotide sequences of the primer pair are shown as SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

10. A method of producing all-male Micropterus salmoides by breeding, characterized in that, Using the detection reagent of any one of claims 2-4 or the method of claim 8 or 9 to detect and screen YY super-male largemouth bass in offspring.

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