Specific marker of intergenic region of oplegnathus fasciatus ccl14 / pnut, primer pair, detection method, kit and application

Abstract

The present application relates to the technical field of molecular biology, in particular to a specific marker of the ccl14 / pnut intergenic region of Oplegnathus fasciatus, a primer pair, a detection method, a kit and application. The specific marker is a nucleotide sequence of the non-insertion and insertion specific marker of the ccl14 / pnut intergenic region DNA fragment of Oplegnathus fasciatus, and the sequence is the base shown in SEQ ID NO: 1 and SEQ ID NO: 2. It is of great significance and application value to carry out female and male gender identification, high-male seed preparation and family selection research based on the ccl14 / pnut intergenic region of Oplegnathus fasciatus.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to specific markers, primer pairs, detection methods, kits, and applications for the intergenic region of the ccl14 / pnut gene in the striped sea bream. Background Technology

[0002] Striped rock sea bream ( Oplegnathus fasciatus The striped rock seabream (Sinonovacula spp.) is an important marine aquaculture and propagation fish in my country, possessing extremely high economic and ornamental value. Its sex determination mechanism is a rare achromosomal system (X1X1X2X2 / X1X2Y), with males carrying a new Y chromosome formed by chromosome fusion, resulting in significantly faster growth than females. This characteristic makes the breeding of all-male or highly male fry a key direction for improving aquaculture efficiency. However, the striped rock seabream lacks external sex characteristics in its juvenile stage, making early and accurate identification of genetic sex impossible using traditional methods, severely hindering the progress of sex-controlled breeding. Therefore, developing molecular markers and rapid PCR identification technologies based on Y chromosome-specific structural variations (such as large DNA fragment insertions) is of vital application value for achieving accurate early genetic sex identification of fry, efficient breeding of high-quality male germplasm, and promoting the improvement of industry quality and efficiency.

[0003] CCL14 (CC motif chemokine ligand 14), a member of the chemokine family, plays a crucial regulatory role in immune responses and inflammatory reactions. As an important biomarker for acute kidney injury (AKI), CCL14 has moderate predictive power for renal function recovery (AUC=0.71), providing a valuable tool for clinical prognosis assessment of AKI patients, particularly suitable for early risk identification in intensive care settings. Pnut (Pnut septin7-like protein) belongs to the septin protein family and is a class of GTP-binding proteins that can form heteromeric complexes. This protein has multiple functions in cell dynamics: participating in cytokinesis, membrane dynamics, and cytoskeleton organization. Pnut's important function is manifested in muscle tissue, where it maintains sarcomere structural integrity and muscle function through interaction with the aptamer protein Numb. To date, there are no reports of using DNA insertion variations in the ccl14 / pnut intergenic region for the genetic identification of male and female striped seabream. 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 intergenic region of the ccl14 / pnut gene in the striped sea bream, and to provide specific markers, primer pairs, detection methods, kits, and applications for the ccl14 / pnut intergenic region of the striped sea bream.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A specific marker for the intergenic region of the ccl14 / pnut gene in the striped sea bream, wherein the specific marker is the nucleotide sequence of the non-insertion and insertion specific markers of the DNA fragment of the ccl14 / pnut intergenic region of the striped sea bream, and the sequence is the base shown in SEQ ID NO:1 and SEQ ID NO:2.

[0007] The nucleotide sequence SEQ ID NO:1 in the non-insertion and insertion-specific marker of the ccl14 / pnut intergenic DNA fragment of the striped rock sea bream is a homologous fragment in the ccl14 / pnut intergenic region shared by the X1 chromosomes of both male and female striped rock sea bream, in which no DNA insertion variation has occurred. It is a common DNA characteristic marker of the ccl14 / pnut intergenic region of the striped rock sea bream, in both non-insertion and insertion individuals.

[0008] The nucleotide sequence SEQ ID NO:2 is a deletion fragment (3bp) between positions 629 and 631 and an insertion fragment (674bp) between positions 702 and 703 of the base sequence shown in SEQ ID NO:1. This means that DNA insertion has occurred in the intergenic region of the ccl14 / pnut gene shared by the X1 chromosomes of both male and female striped rock seabream. The inserted fragment is a marker of individual DNA difference.

[0009] An application of the specific marker described above, in detecting the genetic sex of male and female striped seabream.

[0010] A method for identifying the genetic sex of male and female striped rock seabream using the aforementioned specific marker.

[0011] 1) PCR amplification: Take the striped rock seabream to be tested and extract genomic DNA; use the obtained genomic DNA as a template, use the striped rock seabream ccl14 / pnut intergenic region specific primers to perform PCR amplification, and compare the obtained PCR amplification product with the non-insertion and insertion specific markers of the striped rock seabream ccl14 / pnut intergenic region DNA fragment.

[0012] 2) Result Interpretation: A DNA fragment of only 760 bp was amplified from the genomic DNA of the tested striped rock seabream. This indicates that the tested striped rock seabream is an individual from which no DNA insertion variation has occurred in the ccl14 / pnut intergenic region; this individual is female X1X1X2X. 2；

[0013] Two target bands, 1431bp and 760bp, were amplified, which indicates that the tested striped rock seabream is an individual with a DNA fragment insertion mutation in the intergenic region of the ccl14 / pnut gene; the individual is male X1X2Y.

[0014] The specific primer is

[0015] Ch ccl14 / pnut _F:1 : 5'-TGGACAACTTCACAAGCTGCTAG-3';

[0016] Ch ccl14 / pnut _R:2 : 5'-TCAGATGAAACCTGCCATTATGGC-3'.

[0017] A primer for detecting a specific marker of intergenic insertion variation in the intergenic region of the striped seabream.

[0018] Ch ccl14 / pnut _F:1 : 5'-TGGACAACTTCACAAGCTGCTAG-3';

[0019] Ch ccl14 / pnut _R:2 : 5'-TCAGATGAAACCTGCCATTATGGC-3'.

[0020] A kit for identifying the genetic sex of striped rock seabream, the kit containing the aforementioned primers.

[0021] Advantages of this invention:

[0022] This invention identifies a large DNA sequence insertion in the ccl14 / pnut intergenic region of the male striped rockfish's fusion chromosome Y compared to the ccl14 / pnut intergenic region on the female's homologous chromosome X. This allows for efficient, rapid, and accurate identification of whether DNA fragment insertion variation has occurred in the ccl14 / pnut intergenic region of the striped rockfish. This is of great significance and application value for revealing the differences in immunity and cell dynamics between male and female striped rockfish, achieving rapid identification of the genetic sex of males and females, and improving the genetic breeding process of striped rockfish and the large-scale production of high-quality seedlings.

[0023] This invention screened and obtained homologous regions in the intergenic region of the ccl14 / pnut gene on the X and Y chromosomes of the striped rock seabream, and also identified a large DNA insertion marker sequence between them. Based on this, a rapid detection method for DNA insertion variations in the ccl14 / pnut intergenic region of the striped rock seabream was established. This method can quickly, accurately, and efficiently identify whether DNA sequence insertion variations have occurred in the ccl14 / pnut intergenic region of the striped rock seabream, thereby distinguishing the genetic sex of the seabream. Using a pair of primers, two bands, 1431 bp and 760 bp, differing by 671 bp, were amplified in individuals with DNA sequence insertion variations in the ccl14 / pnut intergenic region. In individuals without DNA fragment insertions in the ccl14 / pnut intergenic region, only a single 760 bp band was amplified, which can be distinguished by agarose gel electrophoresis. This shortens the time for accurate identification of ccl14 / pnut intergenic region variations in the striped rock seabream and improves detection efficiency. This study is of great significance and application value in the research on the regulation of female and male immunity and cell dynamics in striped rock seabream based on intergenic region variation of ccl14 / pnut, as well as in sex identification, preparation of male seedlings and family selection. Attached Figure Description

[0024] Figure 1 This invention provides an embodiment of the nucleotide sequence alignment diagram of the Y chromosome ccl14 / pnut intergenic region, the X chromosome ccl14 / pnut intergenic region, and the ChrX1 chromosome. The modules connected by the lines in the diagram represent highly homologous regions of the ccl14 / pnut intergenic region on the X and Y chromosomes, with the areas enclosed by diagonal lines on the left and right representing the target sequence ChrX1 of this invention. ccl14 / pnut and Chry ccl14 / pnut Location.

[0025] Figure 2 ChrX1 provided for embodiments of the present invention ccl14 / pnut and Chry ccl14 / pnut Specific location information on the X and Y chromosomes; 760bp represents ChrX1. ccl14 / pnut The length of the fragment, 1431bp represents Chrys. ccl14 / pnut Length; Chry ccl14 / pnut The middle low region represents the inserted 671 bp nucleotide sequence, and this region is related to ChrX1. ccl14 / pnut The fragment has no homologous matching sequence.

[0026] Figure 3 The X chromosome ChrX1 obtained as provided in the embodiments of the present invention ccl14 / pnut ChrY with Y chromosome ccl14 / pnut Nucleotide sequence alignment diagram, with primer positions at both ends indicated by double black underlines; *: represents ChrX1 ccl14 / pnutand Chry ccl14 / pnut Sequence consistency: blank areas indicate sequences with inconsistent bases; _ _ _ : represents inserted or missing sequences; black single underline represents the region where the inserted sequence is located.

[0027] Figure 4 The X chromosome ChrX1 provided in the embodiments of the present invention ccl14 / pnut ChrY with Y chromosome ccl14 / pnut 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.

[0028] Figure 5 The image shows the 1.5% agarose gel electrophoresis results of PCR products from male and female striped rockfish provided in this embodiment of the invention. M: DL 2000 DNA Maker; ♀: physiological female fish; ♂: physiological male fish. Individuals showing a single band (760bp) are individuals where no DNA sequence insertion has occurred in the intergenic region of the ccl14 / pnut gene, and are also genetically female. Histological sex identification is female. Individuals showing two bands (1431bp and 760bp) are individuals where DNA sequence insertion has occurred in the intergenic region of the ccl14 / pnut gene, and are also genetically male. Histological sex identification is male. Detailed Implementation

[0029] 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.

[0030] This invention first used third-generation PacBio whole-genome sequencing technology to complete the sequencing and assembly of the whole genomes of female and male striped rockfish, obtaining high-quality genomes at the chromosome level (SRP160016, SRP220007, Xiao et al., 2019, 2020). Comparative genomic bioinformatics analysis revealed a large DNA sequence insertion variation in the homologous ccl14 / pnut intergenic region on the Y chromosome of male striped rockfish, compared to the ccl14 / pnut intergenic region on the X1 chromosome of females. Figure 1 and Figure 2 The target regions were selected as follows: the 30,460,088bp~30,460,847bp region of the ccl14 / pnut gene intergenic region on the X1 chromosome of female fish and the 30,382,543bp~30,383,973bp region of the Y chromosome of male fish that is homologous to the ccl14 / pnut gene and contains DNA sequence insertion. Figure 1 The DNA fragment on chromosome X1 is 760 bp in length and is named ChrX1.ccl14 / pnut Its sequence is the base X shown in SEQ ID NO:1. ccl14 / pnut ID No: 1; The DNA fragment on the Y chromosome is 1431 bp in length and contains a homologous sequence to the intergenic region of ccl14 / pnut on the X1 chromosome, named ChrY. ccl14 / pnut Its sequence is the Y base shown in SEQ ID NO:2. ccl14 / pnut ID No:2; Chry ccl14 / pnut With ChrX1 ccl14 / pnut Homologous sequence alignment revealed that it was related to ChrX1 ccl14 / pnutr Two DNA sequence insertions and deletions, each 671 bp in size, were found between positions 629 and 631 (3 bp deletion) and positions 702 and 703 (674 bp insertion). Figure 3 and Figure 4 In the intergenic region of the male Y chromosome ccl14 / pnut gene, between 30,382,543 bp and 30,383,973 bp, a 674 bp DNA sequence was inserted and a 3 bp deletion was present compared to the homologous region of the ccl14 / pnut intergenic region on the female X1 chromosome. This inserted fragment serves as a DNA marker for detecting DNA insertion variation in the ccl14 / pnut intergenic region of the striped 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 striped rock seabream. ccl14 / pnut This is a DNA characteristic marker shared by males and females of the striped rock seabream.

[0031] This invention uses a long DNA insertion sequence marker found at positions 30,382,543bp~30,383,973bp in the intergenic region of the ccl14 / pnut gene on the Y chromosome of male striped rockfish to determine whether a DNA insertion variation has occurred in the ccl14 / pnut gene region. This marker can then be used for rapid identification of the genetic sex of striped rockfish. Specifically, the detection method involves determining whether the striped rockfish being tested possesses a Y chromosome. ccl14 / pnut The nucleotide fragment (1431 bp) of ID No:2 was rapidly identified using PCR primers. This invention is based on a comparative analysis of the intergenic region sequence of the ccl14 / pnut gene on the Y chromosome of the striped rock seabream and homologous gene sequences on chromosomes X1 and X2, identifying the intergenic region X of the ccl14 / pnut gene on chromosome X1. ccl14 / pnut The Y chromosome with a nucleotide sequence homologous to ID No:1 ccl14 / pnutBased on the nucleotide sequence ID No:2, and utilizing the characteristic of a large insertion of DNA fragments in the homologous region of the ccl14 / pnut gene intergenic region on the Y chromosome of the striped rockfish, 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 ccl14 / pnut gene intergenic region of the tested striped rockfish. 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 striped rockfish. The upstream and downstream primer sequences are as follows:

[0032] Ch ccl14 / pnut _F:1 : 5'-TGGACAACTTCACAAGCTGCTAG-3'(X ccl14 / pnut ID No:2)(SEQ IDNO:3;

[0033] Ch ccl14 / pnut _R:2 : 5'-TCAGATGAAACCTGCCATTATGGC-3'(X ccl14 / pnut ID No:3 (SEQID NO:4).

[0034] The steps for identifying DNA insertion mutations in the intergenic region of the ccl14 / pnut gene in the striped rock seabream using the primers described above mainly include: extracting high-quality whole-genome DNA from the striped rock seabream, amplifying specific marker DNA fragments of the ccl14 / pnut gene intergenic region insertion mutation on the Y chromosome, and detecting the PCR product DNA by agarose gel electrophoresis. Among them, two DNA fragments of 1431 bp and 760 bp were amplified in the Y chromosome and X1 chromosome of the male striped rock seabream (X1X2Y), respectively. The 1431 bp fragment is a marker fragment specific to the ccl14 / pnut gene intergenic region where the DNA insertion mutation occurred. However, only a single DNA fragment of 760 bp was amplified in the female striped rock seabream (X1X1X2X2).

[0035] This invention is based on the whole genome sequencing of male and female striped rockfish and the localization and DNA sequence alignment analysis of the ccl14 / pnut gene intergenic region in male and female individuals. It was found that a long DNA fragment insertion variation occurred in the ccl14 / pnut gene intergenic region on the Y chromosome of male striped rockfish. This DNA fragment insertion variation is used as a unique DNA marker for the ccl14 / pnut gene intergenic region insertion variation on the Y chromosome of striped rockfish. The sex of striped rockfish can be quickly identified by the ccl14 / pnut gene intergenic region DNA sequence insertion variation. This method can quickly, accurately and efficiently distinguish whether the tested striped rockfish has the ccl14 / pnut gene intergenic region sequence insertion variation. This method amplifies two bands, 1431 bp and 760 bp, in individuals of the striped rockfish with DNA insertion in the ccl14 / pnut intergenic region. The 1431 bp band is the specific target band, while in individuals without DNA insertion in the ccl14 / pnut intergenic region, only a single band (760 bp) is amplified. These target bands can be rapidly and accurately distinguished using agarose gel electrophoresis, enabling rapid identification of whether DNA sequence insertion has occurred in the ccl14 / pnut intergenic region of the striped rockfish. Furthermore, since this specific target band (1431 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 the striped rockfish, and can be used for rapid identification of the genetic sex of the striped rockfish.

[0036] Example 1: Screening and Validation of DNA Markers Specific to Intergenic Sequence Variations in the ccl14 / pnut Gene of a Rockfish

[0037] Discovery of a homologous region between the ccl14 / pnut genes on the Y and X chromosomes of the striped rock seabream, containing a large insertion target DNA sequence: The DNA sequences of the male neo-Y chromosome and the female X1 chromosome were obtained from the research team of Li Jun at the Marine Fish Culture and Breeding Technology Laboratory, Institute of Oceanology, Chinese Academy of Sciences. The team commissioned Wuhan Fraser Gene Information Co., Ltd. to use third-generation PacBio whole-genome sequencing technology to complete the sequencing and assembly of the male and female striped rock seabream genomes. The assembly results have been published (SRP160016, SRP220007, Xiao et al., 2019, 2020). Comparative genomic bioinformatics analysis of the male and female striped rock seabream genome sequences shows (see...). Figure 1 and Figure 2 The intergenic region of the ccl14 / pnut gene on the heteromorphic Y chromosome of male striped rockfish is homologous to the intergenic region of the ccl14 / pnut gene on the X1 chromosome of female striped rockfish, and a large DNA insertion fragment is present. The target DNA fragment in the intergenic region of the ccl14 / pnut gene on the X1 chromosome is 760 bp in length and is named ChrX1. ccl14 / pnutIts sequence is SEQ ID NO:1:

[0038] .

[0039] The target DNA fragment homologous to the ccl14 / pnut gene region on the heteromorphic Y chromosome of male fish is 1431 bp in length, and it contains a sequence homologous to X1, named ChrY. ccl14 / pnut Its sequence is SEQ ID NO:2:

[0040]

[0041] Whole genome scans of males and females, and localization and DNA sequence alignment analysis of the ccl14 / pnut intergenic region in males and females, showed that it corresponds to the ChrX1 segment located in the ccl14 / pnut intergenic region on chromosome X1. ccl14 / pnut homologous Chrys ccl14 / pnut Two DNA sequence insertions and deletions occurred on the Y chromosome, corresponding to ChrX1 and ChrX2, respectively. ccl14 / pnut Two DNA sequence insertions and deletions, each 671 bp in size, were found between positions 629 and 631 (deletion of 3 bp) and between positions 702 and 703 (674 bp). Figure 4 The target region between the ccl14 / pnut genes on the Y chromosome (ChrY). ccl14 / pnut DNA sequence ratio with homologous DNA regions of chromosome X1 (ChrX1) ccl14 / pnut The sequence involves an insertion of a 674 bp DNA sequence and a 3 bp deletion. The inserted DNA fragment is a unique DNA marker for the intergenic mutation in the ccl14 / pnut gene region of the striped rockfish. Its presence or absence can be used to identify whether a DNA sequence insertion mutation has occurred in the ccl14 / pnut gene region of the striped rockfish. Furthermore, because this marker is located on the male Y chromosome, it exhibits male-linked inheritance characteristics; therefore, ChrY... ccl14 / pnut This fragment is also a DNA marker unique to the Y chromosome of male striped rockfish, and ChrX1 ccl14 / pnut These are DNA markers shared by males and females of the striped rock seabream, such as... Figure 3 and Figure 4 As shown.

[0042] Sequence verification of DNA markers specific to the ccl14 / pnut intergenic region sequence variation: Based on the characteristics of the SEQ ID NO:2 nucleotide sequence of the ccl14 / pnut intergenic region on the Y chromosome and the SEQ ID NO:1 nucleotide sequence of the homologous gene on the X1 chromosome, two primers were designed ( Figure 4 Selected female and male striped rock seabream of known physiological sex and extracted high-quality DNA from them, while using Ch... ccl14 / pnut _F:1、Ch ccl14 / pnut PCR amplification was performed using two primers, _R:2. The reaction conditions and procedure were as follows: The PCR reaction system was 20 µL, including 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... ccl14 / pnut _F:1 0.4µL; Ch ccl14 / pnut_R:2 0.4 µL; DNA template 2.0 µL, 7.2 µL ddH2O; mix and centrifuge. Touch-down PCR amplification program: 94℃ 3 mins, 63℃ (-1℃, 3 cycles) 1 min, 72℃ 1 min 30 s, 3 cycles; 94℃ 30 s, 60℃ 1 min, 72℃ 1 min 30 s, 30 cycles; 72℃ 10 min, store at 15℃. PCR products were analyzed by 1.5% agarose gel electrophoresis, which revealed differences between individuals in the intergenic region of the ccl14 / pnut gene with and without inserted DNA fragments. The differential fragments in the intergenic region of the ccl14 / pnut gene between inserted and non-inserted DNA sequences were recovered from the gel and transformed into competent cells using the PMD18-T vector. Positive clones were selected for testing. Sequencing results confirmed that the intergenic region of the ccl14 / pnut gene on the X and Y chromosomes of the striped rock seabream contained a large inserted target DNA sequence fragment, such as... Figure 2 and Figure 3 As shown.

[0043] Example 2: Establishment and Application of DNA Fragment Insertion Variation Identification Technology in the Ccl14 / Pnut Intergenic Region of a Rockfish

[0044] Genetic identification was performed on striped rock seabream (12 of which were female and 12 were male) raised by Haihe Aquatic Seedling Co., Ltd. in Wendeng District, Weihai City, Shandong Province:

[0045] High-quality DNA extraction: DNA was extracted from the fin rays of the striped rock seabream 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.

[0046] PCR reaction system and PCR amplification identification: Primers specific to the large sequence variation in the intergenic region of the ccl14 / pnut intergenic region of the striped rock seabream were used. ccl14 / pnut _F:1 and Ch ccl14 / pnut _R:2 Insertion mutation of the intergenic DNA fragment in the ccl14 / pnut gene of the striped sea bream was detected by PCR. The PCR reaction mixture (20 µL) consisted of: 5.8 µL 10× Buffer, 4.0 µL dNTPs, 0.2 µL rTaq enzyme (5 U / µL), and forward and reverse primers (Ch... ccl14 / pnut _F:1 and Ch ccl14 / pnut_R:2) 0.4µL each, DNA template 2.0µL, ddH2O 7.2µL. Touch-down PCR amplification program: 94℃ 3mins, 63℃ (-1℃, 3 cycles) 1min, 72℃ 1min30s, 3 cycles; 94℃ 30s, 60℃ 1min, 72℃ 1min30s, 30 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 30 minutes. Gel imaging can clearly distinguish individuals with DNA insertion and non-insertion variations in the intergenic region of the ccl14 / pnut gene in *Sinocyclocheilus davidii* (see [link to relevant documentation]). Figure 5 ).

[0047] Depend on Figure 5 It is evident that two target bands (1431 bp and 760 bp) are amplified in individuals of the striped rock seabream with DNA insertion in the ccl14 / pnut intergenic region. The 1431 bp band is the ChrY band, which is the specific target band for DNA fragment insertion in the ccl14 / pnut intergenic region. ccl14 / pnut In individuals where DNA fragment insertion into the ccl14 / pnut intergenic region did not occur, only a single band of ChrX1 was amplified. ccl14 / pnut (760bp). Due to ChrY ccl14 / pnut The marker is located on the male Y chromosome and exhibits male-linked inheritance characteristics; therefore, ChrY... ccl14 / pnut This fragment is also a DNA marker unique to the Y chromosome of male striped rockfish, and ChrX1 ccl14 / pnut This method identifies the shared DNA characteristics of male and female striped rock seabream, allowing for the differentiation of selected seabream. The method described in this invention not only enables rapid, accurate, and efficient identification of DNA insertion variations in the ccl14 / pnut intergenic region of the striped rock seabream, but also holds significant importance and application value in the study of male and female immunity and cell dynamics based on the ccl14 / pnut intergenic region, as well as in sex identification, high-male seedling preparation, and family breeding of striped rock seabream.

Claims

1. A specific molecular marker for sex identification of striped seabream, characterized in that: The specific 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. The application of the specific molecular marker according to claim 1 in detecting the genetic sex of striped rock seabream, characterized in that: Specifically 1) PCR amplification: Genomic DNA was extracted from the striped rock seabream to be tested; using the obtained genomic DNA as a template, PCR amplification was performed using primers; 2) Result Interpretation: A DNA fragment of only 760 bp was amplified from the genomic DNA of the tested striped rock seabream, indicating that the tested striped rock seabream was female. ； Two target bands, 1431bp and 760bp, were amplified, indicating that the tested striped rock seabream was male; The primers are Ch ccl14 / pnut _F:1 : 5’-TGGACAACTTCACAAGCTGCTAG-3’; Ch ccl14 / pnut _R:2 : 5'-TCAGATGAAACCTGCCATTATGGC-3'。 3. A method for identifying the genetic sex of striped rock seabream using the specific molecular markers described in claim 1, characterized in that, 1) PCR amplification: Genomic DNA was extracted from the striped rock seabream to be tested; using the obtained genomic DNA as a template, PCR amplification was performed using primers; 2) Result Interpretation: A DNA fragment of only 760 bp was amplified from the genomic DNA of the tested striped rock seabream, indicating that the tested striped rock seabream was female. ； Two target bands, 1431bp and 760bp, were amplified, indicating that the tested striped rock seabream was male; The primers are Ch ccl14 / pnut _F:1 : 5’-TGGACAACTTCACAAGCTGCTAG-3’; Ch ccl14 / pnut _R:2 : 5'-TCAGATGAAACCTGCCATTATGGC-3'。 4. A primer for detecting the specific molecular marker of claim 1, characterized in that: The primers are Ch ccl14 / pnut _F:1 : 5’-TGGACAACTTCACAAGCTGCTAG-3’; Ch ccl14 / pnut _R:2 : 5'-TCAGATGAAACCTGCCATTATGGC-3'。 5. A kit for identifying the genetic sex of striped rock seabream, characterized in that: The kit contains the primers as described in claim 4.

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