Specific marker of orf1 gene of oplegnathus punctatus, primer pair, detection method and application

Abstract

The present application relates to molecular biology technology, specifically to a specific marker of Oplegnathus punctatus or1e1 gene, a primer pair, a detection method and application. The specific marker is a nucleotide sequence of the specific marker of non-insertion and insertion of the DNA fragment of the exon region of the Oplegnathus punctatus or1e1 gene, and the sequence is the base shown in SEQ ID NO: 1 and SEQ ID NO: 2. The present application can only amplify a single band of 167 bp in the individual in which the DNA insertion does not occur in the exon region of the or1e1 gene, and can be distinguished by agarose gel electrophoresis, which can shorten the time for accurate identification of the DNA insertion variation in the exon region of the Oplegnathus punctatus or1e1 gene, and improve the detection efficiency. It has important significance and application value in the research of gender identification, high male seed preparation and family selection based on the exon region of the or1e1 gene of Oplegnathus punctatus.

Description

Technical Field

[0001] This invention relates to molecular biology techniques, specifically to a specific marker, primer pair, detection method, and application of the or1e1 gene of the spotted sea bream. Background Technology

[0002] Spotted sea bream ( Oplegnathus punctatus Rockfish (Stonefish spp.) is a marine aquaculture species with significant development potential, possessing a complex X1X1X2X2 / X1X2Y sex determination system, resulting in significant differences in growth between males and females. Previous studies have found that homologous genes (such as amhr2) in the male and female genomes of rockfish with this sex determination system exhibit abundant structural variations (SVs > 50 bp), such as insertions / deletions or copy number variations of male-specific fragments. These SVs may directly alter gene dosage or the integrity of regulatory elements, becoming key switches for sex differentiation. These large SV fragments can serve as extremely stable and easily detectable molecular markers. Based on this, long-fragment PCR or quantitative PCR detection technologies have been developed, possessing high specificity and strong anti-interference capabilities, enabling the establishment of an efficient genetic sex identification system and providing a reliable and transferable core technology solution for parthenogenesis (all-female) breeding.

[0003] The Or1e1 gene (Olfactory Receptor Family 1 Subfamily E Member 1) belongs to the olfactory receptor gene family, the largest gene family in the genome. Its members are all G protein-coupled receptors (GPCRs) with seven transmembrane domains, responsible for recognizing odor molecules and mediating olfactory signal transduction. This gene is located on human chromosome 17p13.3 and encodes an odor receptor protein. Functionally, or1e1 is involved not only in olfactory perception in the nasal cavity, but its expression is also present in non-olfactory tissues such as the reproductive system. For example, in a comparative transcriptome study of the epididymis of yaks and hybrid cattle, downregulation of or1e1 was identified as a differentially expressed gene potentially associated with hybrid cattle infertility, suggesting a possible role in male reproductive physiology. In recent years, significant progress has been made in the study of the regulatory mechanisms of olfactory receptor genes (including the or1e1 family). For instance, an RNA-mediated symmetry-breaking mechanism has been discovered, whereby OR mRNA itself can enhance its own transcription and suppress other competing OR genes by altering its nuclear architecture, thereby achieving the single-gene selection rule where a single olfactory neuron expresses only one receptor. Furthermore, three-dimensional genomics technology revealed the precise regulatory role of the dynamic connections between OR genes and enhancers during development. To date, there are no reports of using DNA insertion variations in the exon region of the or1e1 gene for the genetic identification of male and female spotted sea bream. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies for detecting DNA insertion variations in the exon region of the or1e1 gene of the spotted sea bream, and to provide a specific marker, primer pair, detection method and application for the or1e1 gene of the spotted sea bream.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A specific marker for the or1e1 gene of the spotted sea bream, wherein the specific marker is the nucleotide sequence of the non-insertion and insertion specific markers of the DNA fragment of the exon region of the or1e1 gene of the spotted sea bream, and the sequence is the base shown in SEQ ID NO:1 and SEQ ID NO:2.

[0006] The nucleotide sequence SEQ ID NO:1 in the specific marker is a homologous fragment of the exon region of the or1e1 gene shared by the X2 chromosomes of both male and female spotted sea bream, in which no DNA insertion variation has occurred. It is a DNA characteristic marker shared by individuals with non-inserted and inserted bases in the exon region of the or1e1 gene of spotted sea bream. The nucleotide sequence SEQ ID NO:2 is an insertion fragment (147bp) between positions 112 and 113 of the base sequence shown in SEQ ID NO:1. This means that DNA insertion has occurred in the exon region of the or1e1 gene shared by the X2 chromosomes of both male and female spotted sea bream. The inserted fragment is a marker of individual difference DNA characteristics.

[0007] An application of the specific marker described above, in detecting the genetic sex of male and female spotted sea bream.

[0008] A method for identifying the genetic sex of spotted sea bream using the aforementioned specific marker. 1) PCR amplification: Take the spotted sea bream to be tested and extract genomic DNA; use the obtained genomic DNA as a template, use the specific primers for the exon region of the spotted sea bream or1e1 gene to perform PCR amplification, and compare the obtained PCR amplification product with the non-insertion and insertion specific markers of the spotted sea bream or1e1 gene exon region DNA fragment. 2) Result Interpretation: A DNA fragment of only 167 bp was amplified from the genomic DNA of the tested spotted sea bream. This indicates that the tested spotted sea bream is an individual in which no DNA insertion variation has occurred in the exon region of the or1e1 gene; this individual is female X1X1X2X. 2； Two target bands, 314bp and 167bp, were amplified from the genomic DNA of the spotted sea bream to be tested, which indicates that the spotted sea bream to be tested is an individual with DNA fragment insertion variation in the exon region of the or1e1 gene of the spotted sea bream; the individual is male X1X2Y.

[0009] The specific primer is Chor1e1 _F:1 : 5'-TCCTCTGTGTTGGCCTATTCACTG-3'; Ch or1e1 _R:2 : 5'- GATGTTCTCTGCCTCCTCC-3'.

[0010] Primers for a specific marker to detect insertional variations in the exon region of the *Scotyledon tomentosa* gene. Ch or1e1 _F:1 : 5'-TCCTCTGTGTTGGCCTATTCACTG-3'; Ch or1e1 _R:2 : 5'- GATGTTCTCTGCCTCCTCC-3'.

[0011] A kit for identifying the genetic sex of spotted sea bream, the kit containing the aforementioned primers.

[0012] Advantages of this invention: This invention reveals that the exon regions of the or1e1 gene on the X and Y chromosomes of the spotted sea bream have homologous segments, and there is also a large DNA insertion marker sequence between them. Therefore, it establishes a rapid, accurate, and efficient method for identifying the genetic sex of the spotted sea bream by DNA fragment insertion variation in the exon region of the or1e1 gene. This method can also reveal the differences in exogenous drug metabolism between male and female spotted sea bream, enabling rapid identification of the genetic sex of males and females. This has significant meaning and application value for improving the genetic breeding process of spotted sea bream and the large-scale production of high-quality seedlings. Attached Figure Description

[0013] Figure 1 The embodiments of this invention provide a nucleotide sequence alignment diagram of the female sex marker or1e1 gene exon and chromosome X2; the modules shown in the diagram represent the female sex marker or1e1 gene exon region and the highly homologous region on chromosome X2, wherein the framed region is the target sequence ChrX2 of this invention. or1e1 Location.

[0014] Figure 2 ChrX2 provided for embodiments of the present invention or1e1 and Chry or1e1 Position information of homologous sequence alignment; 167bp represents ChrX2. or1e1 The length of the fragment, 314bp represents Chrys. or1e1 Length; Chry or1e1 The middle low region represents the inserted 147bp nucleotide sequence, and this region is related to ChrX2. or1e1 The fragment has no homologous matching sequence.

[0015] Figure 3 The obtained X2 chromosome ChrX2 provided in the embodiments of the present invention or1e1 ChrY with Y chromosome or1e1 Nucleotide sequence alignment diagram, with the positions of the primers at both ends indicated by double black underlines; *: represents ChrX2 or1e1 and Chry or1e1 Sequence consistency: blank areas indicate sequences with inconsistent bases; ------: represents insertion or deletion sequences; black single underline represents the region where the insertion sequence is located.

[0016] Figure 4 The X chromosome ChrX2 provided in the embodiments of the present invention or1e1 ChrY with Y chromosome or1e1 A schematic diagram of DNA fragments with homologous insertions / deletions in nucleotide sequences; black areas represent homologous regions, blank areas represent deleted regions and site information, and gray areas represent inserted regions and site information.

[0017] Figure 5 The image shows the 1.5% agarose gel electrophoresis results of PCR products from male and female spotted sea bream provided in this embodiment of the invention. M: DL 2000 DNA Maker; ♀: physiological female fish; ♂: physiological male fish. Individuals showing a single band (167bp) are individuals where no DNA sequence insertion has occurred in the exon region of the or1e1 gene, and are also genetically female, and are physiologically sexed by histological identification as female. Individuals showing two bands (314bp and 167bp) are individuals where DNA sequence insertion has occurred in the exon region of the or1e1 gene, and are also genetically male, and are physiologically sexed by histological identification as male fish. Detailed Implementation

[0018] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0019] This invention utilizes the published genome information of the spotted sea bream (CNP0001488, Li et al., 2021); through comparative genomic bioinformatics analysis, it was discovered that compared to the exon region of the or1e1 gene on the X2 chromosome of female spotted sea bream, a large DNA sequence insertion variation occurred in the exon region of the homologous or1e1 gene on the Y chromosome of male spotted sea bream. Figure 1 and Figure 2 The study targeted regions were selected from the exon region 33,786,481bp~33,786,647bp of the or1e1 gene on the female X2 chromosome and the homologous region 33,857,877bp~33,858,190bp of the male Y chromosome containing DNA sequence insertions. Figure 1 The DNA fragment on chromosome X2 is 167 bp in length and is named ChrX2. or1e1 Its sequence is SEQ ID NO:1 (X or1e1 The bases shown in ID No:1; the DNA fragment on the Y chromosome is 314 bp in length and contains a sequence homologous to the exon region of the or1e1 gene on the X2 chromosome, named ChrY. or1e1 Its sequence is SEQ ID NO:2 (Y or1e1 The bases shown in ID No:2; ChryY or1e1 With ChrX2 or1e1 Homologous sequence alignment revealed that it was related to ChrX2 or1e1r An insertion of a DNA sequence of 147 bp was found between positions 112 and 113 (147 bp insertion). Figure 3 and Figure 4 In the male Y chromosome, the exon region of the or1e1 gene contains a 147 bp DNA sequence inserted into the homologous region of the or1e1 gene exon region on the female X2 chromosome, specifically in the area from 33,857,877 bp to 33,858,190 bp. This inserted fragment serves as a DNA marker for detecting DNA insertion variation in the or1e1 gene exon region of the spotted rock seabream. Furthermore, because this marker is located on the male Y chromosome and exhibits male-linked inheritance characteristics, this fragment is also a DNA marker unique to the male Y chromosome of the spotted rock seabream. or1e1 It contains DNA markers that are shared by male and female spotted sea bream for sex.

[0020] This invention uses a long DNA insertion sequence marker found at positions 33,857,877bp~33,858,190bp in the exon region of the or1e1 gene on the Y chromosome of male spotted sea bream to determine whether a DNA insertion variation has occurred in the exon region of the or1e1 gene. This marker can then be used for rapid identification of the genetic sex of spotted sea bream. Specifically, the detection method involves determining whether the spotted sea bream being tested possesses a Y chromosome. or1e1 The nucleotide fragment (314 bp) of ID No:2 was rapidly identified using PCR primers. This invention is based on a comparative analysis of the exon region sequence of the or1e1 gene on the Y chromosome of the spotted sea bream and homologous gene sequences on chromosomes X1 and X2, determining the sequence corresponding to the or1e1 gene exon region X on chromosome X2. or1e1 The Y chromosome with a nucleotide sequence homologous to ID No:1 or1e1Based on the nucleotide sequence ID No:2, and utilizing the characteristic of a large insertion of DNA fragments into the homologous region of the or1e1 gene exon on the Y chromosome of the spotted sea bream, two primers were designed. The PCR products were detected by 1.5% agarose gel electrophoresis to quickly determine whether DNA fragment insertion variation had occurred in the exon region of the or1e1 gene in the tested spotted sea bream. Simultaneously, utilizing the fact that this marker is located on the male Y chromosome and has a male-linked inheritance characteristic, it can be applied to the accurate identification of the genetic sex of the spotted sea bream. The upstream and downstream primer sequences are as follows: Ch or1e1 _F:1 : 5'-TCTCTGTGTTGGCCTATTCACTG-3'(X or1e1 ID No:2)(SEQ ID NO:3; Ch or1e1 _R:2 : 5'- GATGTTCTCTGCCTCCTCC-3'(X or1e1 ID No:3 (SEQ ID NO:4).

[0021] The steps for identifying DNA insertion mutations in the exon region of the or1e1 gene of the spotted rock seabream using the above primers mainly include: extracting high-quality whole-genome DNA of the spotted rock seabream, amplifying specific marker DNA fragments of DNA insertion mutations in the exon region of the or1e1 gene on the Y chromosome, and detecting the DNA of the PCR product by agarose gel electrophoresis; among them, two DNA fragments of 314bp and 167bp were amplified in the Y chromosome and X2 chromosome of male spotted rock seabream (X1X2Y), respectively. The 314bp fragment is a marker fragment specific to the exon region of the or1e1 gene where DNA insertion mutations have occurred; while in female spotted rock seabream (X1X1X2X2) individuals, only a single DNA fragment of 167bp was amplified.

[0022] This invention is based on the whole genome sequencing of male and female spotted sea bream and the localization and DNA sequence alignment analysis of the exon region of the or1e1 gene in male and female individuals. It can be seen that a long DNA fragment insertion variation has occurred in the exon region of the or1e1 gene on the Y chromosome of male spotted sea bream. This can be used as a DNA marker for the base insertion variation in the exon region of the or1e1 gene on the Y chromosome of spotted sea bream. The sex of spotted sea bream can be quickly identified by the DNA sequence insertion variation in the exon region of the or1e1 gene. This method can quickly, accurately and efficiently distinguish whether the tested spotted sea bream has the sequence insertion variation in the exon region of the or1e1 gene. This method amplifies two bands, 314 bp and 167 bp, in individuals of the spotted rockfish with DNA insertion in the exon region of the or1e1 gene. The 314 bp band is the specific target band, while only a single band (167 bp) is amplified in individuals without DNA insertion in the exon region of the or1e1 gene. These target bands can be quickly and accurately distinguished using agarose gel electrophoresis, enabling rapid identification of whether DNA sequence insertion has occurred in the exon region of the or1e1 gene in spotted rockfish. Furthermore, since this specific target band (314 bp) is located on the male Y chromosome and exhibits male-linked inheritance characteristics, this fragment is also a DNA marker unique to the male Y chromosome of spotted rockfish, and can be used for rapid identification of the genetic sex of spotted rockfish.

[0023] Example 1: Screening and Validation of DNA Markers Specific to Exon Sequence Variations in the Or1e1 Gene of the Spotted Rockfish Discovery of homologous regions in the exons of the or1e1 gene on the Y and X chromosomes of the spotted rock snapper, containing large insert target DNA sequences: The male neo-Y chromosome DNA sequence and the female X2 chromosome DNA sequence were derived from published spotted rock snapper genome information (CNP0001488, Li et al., 2021). Comparative genomic bioinformatics analysis of the male and female spotted rock snapper genome sequences reveals (see...) Figure 1 and Figure 2 The exon region of the or1e1 gene on the heteromorphic Y chromosome of male spotted sea bream is homologous to the exon region of the or1e1 gene on the X2 chromosome of female spotted sea bream, and a large DNA insertion fragment is present. The target DNA fragment in the exon region of the or1e1 gene on the X2 chromosome is 167 bp in length and is named ChrX2. or1e1 Its sequence is SEQ ID NO:1: TCCTCTGTGTTGGCCTATTCACTGAAGAGTCTGGACGCTCAGCCTGACGTTCTGCCTCCTGAACAGACGGCAGAAATTAGTAAGGCTTCGAGCGCCATCCTGACCCTGTGTGTTTTCCTGGAAATGGGGCATGATCTAAAGTCCCGCGTCGGAGGAGGCAGAGAACATC.

[0024] The target DNA fragment homologous to the exon region of the or1e1 gene on the heteromorphic Y chromosome of male fish is 314 bp in length, and it contains a sequence homologous to X2, named ChrY. or1e1 Its sequence is SEQ ID NO:2: TCCTCTGTGTTGGCCTATTCACTGAGGAGTCTGGCCGCTCAGCCTGACGTTTTGCCTCCTGAACAGGCGGCAGAAATTAGTAAGGCTTTGAGCGCCATCCTGACCCTGTGTGCATCCGTCGCAGTCTGCTCTGCTAGAATTGCAGCCTGGCAGACTGT GATGCATAGGAACATTTGGCTGCACCTTTCGTCCATTCCTGAGGAGCACAGGAAAGAGCTCCTGGAGGCCCCCATCAGCCTCGACGGGCTGTTCGGGCCCCATTTCCAGGAAATGGTACATGATCTAAAGTCCAAGTTGGAGGAGGCAGAGAACATC.

[0025] Whole genome scans of males and females, along with localization and DNA sequence alignment of the or1e1 gene exon region in both individuals, showed that it corresponds to the ChrX2 segment located in the or1e1 gene exon region on chromosome X2. or1e1 homologous Chry or1e1 A DNA sequence insertion occurred on the Y chromosome, corresponding to ChrX2. or1e1 A 147bp DNA sequence insertion was found between positions 112 and 113 (147bp). Figure 4 The target region of the or1e1 gene exon on the Y chromosome (ChrY). or1e1 DNA sequence ratio with homologous DNA regions of chromosome X2 (ChrX2) or1e1A 147bp DNA sequence is inserted, and this inserted DNA sequence fragment is a unique DNA marker for base insertion variation in the exon region of the *Or1e1* gene in *Syngonium spp.* Its presence or absence can be used to identify whether DNA sequence insertion variation has occurred in the exon region of the *Or1e1* gene in *Syngonium spp.* Furthermore, because this marker is located on the male Y chromosome, it exhibits male-linked inheritance characteristics; therefore, ChrY... or1e1 This fragment is also a DNA marker unique to the Y chromosome of male spotted rock snapper, and ChrX2 or1e1 These are DNA markers shared by males and females of the spotted rock seabream, such as... Figure 3 and Figure 4 As shown.

[0026] Sequence verification of DNA markers specific to sequence variations in the exon region of the or1e1 gene: Based on the characteristics of the SEQ ID NO:2 nucleotide sequence of the or1e1 gene exon region on the Y chromosome and the SEQ ID NO:1 nucleotide sequence of the homologous gene on the X2 chromosome, two primers were designed ( Figure 4 High-quality DNA was extracted from female and male spotted sea bream of known physiological sex, and primers Ch were used. or1e1 _F:1 and Ch or1e1 PCR amplification was performed using _R:2. The specific reaction conditions and procedure are as follows: The PCR reaction system was 20 µL, containing 5.8 µL of 10× Buffer; 4.0 µL of dNTPs (2.5 mmol / L); 0.2 µL of rTaq enzyme (5 U / µL); and Ch... or1e1 _F:1 0.4µL; Ch or1e1 _R: 2 0.4µL; DNA template 2.0µL, 7.2µL ddH2O. After mixing, centrifuge thoroughly. The touch-down PCR program was: 95℃ for 3 min, 58.5℃ (decreasing by 1℃ per cycle, for a total of 3 cycles) annealing for 30 s, 72℃ extension for 1 min, 3 cycles; then 95℃ denaturation for 30 s, 56.5℃ annealing for 30 s, 72℃ extension for 1 min, 28 cycles; finally, 72℃ extension for 10 min, and storage at 15℃. PCR products were analyzed by 1.5% agarose gel electrophoresis to distinguish individual differences between inserted and non-inserted DNA fragments in the exon region of the or1e1 gene. Differential fragment products were recovered by gel extraction and transformed into competent cells using the PMD18-T vector. Positive clones were selected and sent to Qingdao Paisennuo Gene Biotechnology Co., Ltd. for sequencing. Sequencing results confirmed that the or1e1 gene exon region of the X and Y chromosomes of the spotted sea bream contained a target DNA sequence fragment with a large insertion, such as... Figure 2 and Figure 3 As shown.

[0027] Example 2: Establishment and Application of DNA Fragment Insertion Variation Identification Technology in the Exon Region of the Or1e1 Gene of the Spotted Rockfish Genetic identification was performed on spotted sea bream (12 females and 12 males) raised by Laizhou Mingbo Aquatic Products Co., Ltd. in Laizhou City, Yantai, Shandong Province: High-quality DNA extraction: DNA was extracted from the fin rays of the spotted sea bream using the Tiangen Marine Animal DNA Extraction Kit. The integrity of the genomic DNA was identified by 1.0% agarose gel electrophoresis. The OD value of the DNA supernatant was measured using a UV spectrophotometer. The DNA concentration was adjusted to 60 ng / µL and stored at -20℃ for later use.

[0028] PCR reaction system and PCR amplification identification: Primers specific to large sequence variations in the exon region of the or1e1 gene of the spotted sea bream were used. or1e1 _F:1 and Ch or1e1 _R:2 Insertion mutations in the exon region of the or1e1 gene of the spotted sea bream were detected by PCR. The PCR reaction mixture (20 µL) consisted of: 5.8 µL of 10× Buffer, 4.0 µL of dNTPs, 0.2 µL of rTaq enzyme (5 U / µL), and forward and reverse primers (Ch... or1e1 _F:1 and Ch or1e1 _R:2) 0.4µL each, DNA template 2.0µL, ddH2O 7.2µL. Touchdown PCR amplification program: 95℃ 3mins, 58.5℃ (-1℃, 3 cycles) 30s, 72℃ 1min, 3 cycles; 95℃ 30s, 56.5℃ 30s, 72℃ 1min30s, 28 cycles; 72℃ 10min, 15℃ storage. Add 10×Loading Buffer 2.0µL to each PCR sample to be tested, and perform 1.5% agarose gel electrophoresis at a constant voltage of 110V for 30min. Gel imaging can clearly distinguish individuals with DNA insertion and non-insertion variants in the exon region of the or1e1 gene of the spotted sea bream (see...). Figure 5 ).

[0029] Depend on Figure 5 It is evident that two target bands (314 bp and 167 bp) are amplified in individuals of the spotted rock seabream with DNA insertion in the exon region of the or1e1 gene. The 314 bp band is the ChrY band, which is the specific target band for DNA fragment insertion in the exon region of the or1e1 gene. or1e1 In individuals where DNA fragment insertion into the or1e1 gene exon region has not occurred, only a single band of ChrX2 can be amplified. or1e1 (167bp). Due to ChrY or1e1The marker is located on the male Y chromosome and exhibits male-linked inheritance characteristics; therefore, ChrY... or1e1 This fragment is also a DNA marker unique to the Y chromosome of male spotted rock snapper, and ChrX2 or1e1 This method identifies the shared DNA characteristics of male and female spotted sea bream, allowing for the differentiation of selected sea bream. The method of this invention can not only quickly, accurately, and efficiently identify whether DNA fragment insertion variations have occurred in the exon region of the or1e1 gene of the spotted sea bream to be tested, but also has important significance and application value in the study of exogenous drug metabolism in male and female spotted sea bream based on the exon region of the or1e1 gene, as well as in sex identification, high-male seedling preparation, and family selection.

Claims

1. A specific molecular marker for sex identification of spotted sea bream, characterized in that: The molecular markers are DNA fragments with nucleotide sequences as shown in SEQ ID NO:1 and DNA fragments with nucleotide sequences as shown in SEQ ID NO:

2.

2. An application of the specific molecular marker according to claim 1, characterized in that: Application of the specific molecular markers in detecting the genetic sex of spotted sea bream; Specifically: Genomic DNA was extracted from the spotted sea bream to be tested. Using the obtained genomic DNA as a template, PCR amplification was performed using primers, the nucleotide sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:

4. If only a single DNA fragment of 167 bp shown in SEQ ID NO:1 is amplified, the tested spotted sea bream is determined to be female; The amplification of two DNA fragments, 167bp and 314bp, shown in SEQ ID NO:1 and SEQ ID NO:2, indicates that the tested spotted sea bream is male.

3. A primer for detecting the specific molecular marker of claim 1, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:

4.

4. A method for identifying the genetic sex of spotted sea bream using the specific molecular marker described in claim 1, characterized in that, Genomic DNA was extracted from the spotted sea bream to be tested. Using the obtained genomic DNA as a template, PCR amplification was performed using primers, the nucleotide sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:

4. If only a single DNA fragment of 167 bp shown in SEQ ID NO:1 is amplified, the tested spotted sea bream is determined to be female; The amplification of two DNA fragments, 167bp and 314bp, shown in SEQ ID NO:1 and SEQ ID NO:2, indicates that the tested spotted sea bream is male.

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