CN112029843A - Specific molecular marker for identifying genetic sex of scatophagus argus and primers and application thereof - Google Patents
Specific molecular marker for identifying genetic sex of scatophagus argus and primers and application thereof Download PDFInfo
- Publication number
- CN112029843A CN112029843A CN202010986812.3A CN202010986812A CN112029843A CN 112029843 A CN112029843 A CN 112029843A CN 202010986812 A CN202010986812 A CN 202010986812A CN 112029843 A CN112029843 A CN 112029843A
- Authority
- CN
- China
- Prior art keywords
- scatophagus argus
- sex
- chromosome
- molecular marker
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a specific molecular marker for identifying the genetic sex of a scatophagus argus, a primer and application thereof, wherein the molecular marker is two DNA fragments which are partially homologous in an X chromosome and a Y chromosome of the scatophagus argus, the nucleotide sequence of the partially homologous DNA fragment on the X chromosome is shown as SEQ ID NO.1, and the nucleotide sequence of the partially homologous DNA fragment on the Y chromosome is shown as SEQ ID NO. 2. According to the method, two partial homologous DNA fragments on the X chromosome and the Y chromosome of the scatophagus argus are screened from genomic information of the scatophagus argus, and specific primers are designed to identify the genetic sex of the scatophagus argus, so that the genetic sex of the scatophagus argus can be rapidly and accurately distinguished. The method is suitable for accurately identifying the genetic sex of the scatophagus argus in a laboratory or an aquaculture enterprise, can also be used for identifying and screening the sex of the scatophagus argus in different growth stages and different groups, and has important significance for researching sex determination and differentiation mechanisms of the scatophagus argus and realizing sex control of the scatophagus argus.
Description
Technical Field
The invention belongs to the field of biotechnology. More particularly, relates to a specific molecular marker for identifying the genetic sex of the scatophagus argus, a primer and application thereof.
Background
Sex determination, differentiation mechanism and sex control technology breeding of fishes are significant in aquaculture research and application, and many fishes have sex bimorph. If some fishes have sex growth diamorphy, the efficiency can be improved by the unisexual culture. The growth speed of females such as Cynoglossus semilaevis (Chensong forest, etc. 2013) and carp (Cyprinus carpio) (Jiang et al,2020) is obviously faster than that of males, and full-female culture is one of the key technologies for improving the culture yield; males of nile tilapia (Oreochromis niloticus) (Chen et al, 2017) and Pelteobagrus fulvidraco (Dan et al, 2018) are significantly superior to females in growth, and are fully-male-cultured to increase yield and benefit. Some fish species have significant value for a certain sex, e.g. sturgeon roe can produce caviar, so female fish have a higher economic value than male fish (east et al, 2015). Therefore, according to the sex difference of the fish sex economic traits, the development of sex control breeding has important value.
Pseudosciaena crocea (Scatophagus argus) is a medium-small size fish in the euryhaline subtropical zone, and has omnivory, and the minimum maturation age of female and male fish is 1 year, and is generally 2 years. The group breeding period is 4-8 months, 5-7 months are full, and the breeding in seawater pond or net cage can be on the market after one year. The scatophagus argus has delicious taste and tender meat quality, has wide markets in coastal areas, southeast Asia and the like of China, and the current market price reaches 80-120 yuan/kg. The scatophagus argus has obvious male and female growth difference, under the condition of artificial cultivation, the growth speed of 1-year female fish is about 30% faster than that of male fish, and the growth speed of 2-year female fish is more than 100% higher than that of the male fish of the same age (Chuazhen et al, 2010). Therefore, the cultivation efficiency of the whole female cultivation of the scatophagus argus can be obviously improved. The scatophagus argus is also a favorite ornamental fish, the male is more gorgeous than the female, and the all-male scatophagus argus has higher ornamental value, but no unisexual fry breeding report is seen at present. To realize sex control, basic theoretical research on sex-determining molecular mechanism needs to be developed. Due to the plasticity of the sex of the fishes, theoretically, the individuals with genetic sex XX can be induced to develop into pseudo-male fishes (genetically still XX) through the treatment of androgenic steroid hormones, anti-female drugs and aromatase (estrogen synthesis key enzyme) inhibitors, the XX pseudo-male fishes and normal XX female fishes are mated and bred, and finally, all-female offspring is obtained, and the all-female unisexual offspring seed cultivation of the scatophagus argus is realized. Similarly, XY individuals can be induced to develop pseudo female fish (which is still genetically XY) through estrogen treatment, the XY pseudo female fish is mated with XY normal male fish to obtain one fourth of YY super male fish, and the YY super male fish is mated with the normal female fish, so that the cultivation of all-male offspring seeds for viewing and admiring can be realized.
The traditional fish sex control breeding method judges the genotype of a parent through test cross, is time-consuming and labor-consuming, can simply and quickly realize genetic sex based on a sex-linked molecular marker, greatly saves manpower and material resources, and has the defects of high detection cost and long time consumption because the marker needs to perform Sanger sequencing on a PCR amplification product although a specific molecular marker related to the genetic sex of the scatophagus argus is published.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a specific molecular marker for identifying the genetic sex of a scatophagus argus, a second object of the present invention is to provide an application of the specific molecular marker in the preparation of primers for identifying the genetic sex of the scatophagus argus, a third object of the present invention is to provide a pair of primers capable of amplifying the specific molecular marker or a partial fragment thereof by PCR, a fourth object of the present invention is to provide an application of the specific molecular marker or the primers in the identification of the genetic sex of the scatophagus argus, a fifth object of the present invention is to provide an application of the specific molecular marker or the primers in the preparation of a kit for identifying the genetic sex of the scatophagus argus, a sixth object of the present invention is to provide a method for rapidly identifying the genetic sex of the scatophagus argus, and a seventh.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a specific molecular marker for identifying the genetic sex of the scatophagus argus is two DNA fragments which are partially homologous in X chromosome and Y chromosome of the scatophagus argus, wherein the nucleotide sequence of the DNA fragment which is partially homologous on the X chromosome is shown as SEQ ID NO: 1, the nucleotide sequence of the DNA fragment with partial homology on the Y chromosome is shown as SEQ ID NO: 2, respectively.
2. The specific molecular marker is applied to the preparation of primers for identifying the sex of the scatophagus argus.
3. A pair of primers capable of PCR amplifying the specific molecular marker or partial fragment thereof, wherein PCR amplification products have obvious difference on an X chromosome and a Y chromosome.
As one of the preferable technical schemes, the nucleotide sequence of the forward primer in the primer is shown as SEQ ID NO: 3, the nucleotide sequence of the reverse primer is shown as SEQ ID NO: 4, respectively.
4. The specific molecular marker or the primer is applied to the preparation and identification of the sex of the scatophagus argus.
5. The specific molecular marker or the primer is applied to the preparation of a kit for identifying the sex of the scatophagus argus.
6. A method for rapidly identifying the genetic sex of a scatophagus argus comprises the following steps:
s1, extracting genome DNA of a sample to be detected;
s2, designing a primer, and carrying out PCR amplification on the specific molecular marker or a partial fragment thereof by using the genomic DNA of S1 as a template to obtain a PCR amplification product;
s3, carrying out agarose gel electrophoresis on the PCR amplification product of S2;
s4, analyzing a sequencing result, and judging the genetic sex of the scatophagus argus according to an electrophoresis result, wherein the judgment standard is as follows:
if the PCR amplification product only has a clear band, the sample to be detected is a female fish;
and if the PCR amplification product has two clear bands, the sample to be detected is the male fish.
As one of the preferable technical scheme, the PCR amplification system is 2 XPCR mix 25 uL, 10 umol/L forward and reverse primers are 1 uL respectively, genome DNA is 2 uL, ddH2O21. mu.L, 50. mu.L total.
As one of the preferred technical schemes, the PCR amplification program comprises the following steps: 3min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃ and 1min at 72 ℃ for 37 cycles; extension was carried out at 72 ℃ for 10 min.
7. A kit for identifying the genetic sex of the scatophagus argus comprises the specific molecular marker primer.
The invention has the beneficial effects that:
according to the method, two partial homologous DNA fragments on the X chromosome and the Y chromosome of the scatophagus argus are screened from genomic information of the scatophagus argus, and specific primers are designed to identify the genetic sex of the scatophagus argus, so that the genetic sex of the scatophagus argus can be rapidly and accurately distinguished. The method can amplify specific DNA fragments in male and female individuals, can be distinguished by an agarose gel electrophoresis method, and is suitable for accurately identifying the genetic sex of the scatophagus argus in laboratories or aquaculture enterprises. The method for detecting the genetic sex of the scatophagus argus can also be used for identifying and screening the sex of the scatophagus argus in different growth stages and different groups, and has important significance for researching sex determination and differentiation mechanisms of the scatophagus argus and realizing sex control of the scatophagus argus.
Drawings
FIG. 1 is a technical route of molecular marker-assisted breeding for producing genetic hologynic fish. A is a technical route chart for constructing the production of the all-female fish by the XY system, and B is a technical route chart for constructing the production of the all-male fish by the XY system. E2, estradiol; 17 α -MT, 17 α -methyltestosterone; le, letrozole, aromatase inhibitors.
FIG. 2 is a sequence alignment chart of homologous DNA fragments of X chromosome and Y chromosome of the scatophagus argus. Primer positions are indicated by arrows; the method comprises the following steps: this patent identifies DNA fragment sequences of genetic sex that are deleted from the X chromosome relative to the Y chromosome; black background consensus sequences of X-chromosome and Y-chromosome DNA fragments; -: deletion of the sequence.
FIG. 3 is the agarose gel electrophoresis of PCR products of male and female scatophagus argus in 3 different groups according to the invention. Male parent: the phenotypic sex is female; the method comprises the following steps: the phenotypic sex was male.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1
Obtaining sex-specific markers for scatophagus argus
1. Discovery of sex-specific molecular markers
Second-generation genome surfey sequencing of DNA of muscle tissues of 1-female and 1-male scatophagus argus and transcriptome sequencing of gonad tissues of 3-female and 3-male scatophagus argus, and analysis of sequencing data revealed that the scatophagus argus sex-determining candidate gene dmrt 1. Through analysis of individual re-sequencing data of male and female, the sequence of the dmrt1 gene is only found in the male fish genome, and is not found in the female fish genome, the truncated mutant gene dmrt1b of the male and female fish exists, and by comparing the gene structures of the dmrt1 and the dmrt1b, the similarity between the 5 'to 3' UTR region of the exon of the dmrt1 and the corresponding region on the contig 00204 of the gene dmrt1b is found to be 78.2%, and 1 pair of primers is designed aiming at the gene sequence of the region.
Upstream primer F (SEQ ID NO. 3): 5'-TCAGAGCACAATTGTTAGGCAAAGTGAAC-3', respectively;
downstream primer R (SEQ ID NO. 4): 5'-TCGCTACTTTCACCAATACAGCATGA-3');
the PCR amplification of 10-tailed female scatophagus argus and 10-tailed male scatophagus argus genome DNA is carried out by the pair of primers, a band with the length of about 600bp can be amplified in XX female and XY male individuals, and a band with the length of about 700bp is also amplified in XY individuals.
2. Cloning and sequence analysis of sex-specific molecular markers
Amplifying the genome of 3 female and 3 male scatophagus argus by using the primers, performing agarose gel electrophoresis, respectively cutting gel, recovering a single band amplified from an XX female individual and two bands amplified from an XY male individual, connecting the bands to a pEasy-T3 vector, then transforming to an escherichia coli competent cell, culturing for 1 hour by using an LB liquid culture medium, absorbing 50-100ul of bacterial liquid, coating the bacterial liquid on an LA solid culture medium, culturing overnight, picking out a single bacterial colony, identifying a positive clone by using a PCR method, and sequencing.
The sequencing sequences of 5-10 positive clones from each fish are subjected to multiple alignment by using DNAMAN 4.0 software, and as a result, a single amplification band from female individuals is found to be 593bp, the homology of the sequence among different female individuals is as high as 99.3% -100%, and the sequence is named as SEQ chrX (SEQ ID NO. 1). The length of the fragment from the male individual, which is equal to the length of the amplified band of the female individual, is also 593bp, and the homology among different male individuals is as high as 99.5% -100.0%, so the fragment is also named as SEQ chrX; the segment from male individual larger than the SEQ chrX sequence is 693bp in length, and the sequence homology among different male individuals is as high as 99.7% -100.0%, so the male specific segment is named as SEQ chrY (SEQ ID NO. 2). The homology of the SEQ chrX and SEQ chrY sequences from male individuals is only 72.8% -73.7%, and their homology to the SEQ chrX sequence from female individuals is on average 99.5% and 72.6%, respectively. The results show that the SEQ chrX exists in both male and female individuals, and the SEQ chrY exists only in male individuals, namely the SEQ chrX and the SEQ chrY are from homologous sequences of X chromosomes and Y chromosomes respectively, and the technical route for producing genetic all-female fish by molecular marker-assisted breeding is shown in figure 1.
And (3) carrying out sequence comparison analysis on the X chromosome specific sequence SEQ chrX and the Y chromosome specific sequence SEQ chrY obtained by sequencing. The results are shown in fig. 2, where the sequences of SEQ chrX and SEQ chrY differ mainly by 12: compared with the specific molecular marker of the Y chromosome, the specific molecular marker of the X chromosome is as follows: (1) the 17bp sequence is deleted at 66 bp; (2) the 4bp sequence is deleted at 172 bp; (3) 27bp sequence is deleted at 209 bp; (4) 1bp is inserted at 287; (5) 5bp sequences are deleted at 350bp positions; (6) 3bp sequences are deleted at 391 bp; (7) the 4bp sequence is deleted at 414 bp; (8) the 1bp sequence is deleted at 445 bp; (9) the 9bp sequence is deleted at 455 bp; (10) the 484bp position lacks a 6bp sequence; (11) the 21bp sequence is deleted at 513 bp; (12) the 4bp sequence was deleted at 548 bp.
Example 2
Application of primer sequence for genetic sex identification of scatophagus argus
The method for rapidly and accurately identifying the genetic sex of the scatophagus argus comprises the following steps:
(1) collection and phenotypic sex determination of scatophagus argus from different geographical sources
To verify the accuracy of the patent in genetic sex identification, 213-tailed scatophagus argus (106-tailed phenotypic female fish and 107-tailed phenotypic male fish) in Zhanjiang city, North sea city and Zhuhai city are collected, the phenotypic sex of the scatophagus argus is determined by the appearance of gonads, the ovaries of the female fish are triangulated, and the spermary of the male fish is vesicular.
(2) Extraction of genomic DNA from scatophagus argus
Cutting the tail fin of the scatophagus argus to be detected, and extracting the genomic DNA of the scatophagus argus according to the steps by adopting a genomic DNA extraction kit.
(3) Sequencing detection of DNA of sample to be detected
PCR amplification of the extracted genomic DNA was carried out using SEQ ID NO.3 and SEQ ID NO.4 in example 1;
and (3) PCR reaction system: 2 XPCR mix Buffer 25 uL, 10 umol/L upstream and downstream primers 1 uL each, template DNA2 uL, ddH2O21. mu.L, mixed and centrifuged.
PCR amplification procedure: 3min at 94 ℃; 30s at 94 ℃, 30s at 60 ℃ and 1min at 72 ℃ for 37 cycles; preserving at 72 deg.C for 10min and 4 deg.C.
The PCR product was electrophoresed in 1.0% agarose at 120V for 20 min.
Meanwhile, the genetic sex identification is carried out by using the Goldfish genetic sex Marker Dmrt 3-Marker-F/rt 3-Marker-R in the reference (Mustapha, U.F.; Jiang, D.N.; Liang, Z.H.; Gu, H.T.; Yang, W.; Chen, H.P.; Deng, S.P.; Wu, T.L.; Tian, C.X.; Zhu, C.H.; et al, Male-specific Dmrt1 is a candied site x determination gene in a spotted scat, Aquaculture.
As shown in fig. 3 and table 1, the electrophoretic results of the female fish were single band, the electrophoretic results of the male fish were clear two bands (fig. 3), and the results of the phenotypic identifications of 106-tailed female fish and 107-tailed male fish were subjected to matching analysis, which revealed that the genotypic sex and the phenotypic sex identification of the female fish and the male fish were completely matched, and the matching rate of the genetic sex and the phenotypic sex was 100% (table 1). The results of the genetic sex determination using the molecular Marker Dmrt3-Marker-F/Dmrt3-Marker-R are completely consistent with the results of the present invention (Table 1). From the above results, it can be seen that the sex-specific molecular marker of the present invention is suitable for use in a plurality of scatophagus argus from different geographical sources, and has universality.
TABLE 13 genetic sex determination results for Christina loosestrife from different geographical sources
Note: male parent: the phenotypic sex is female; the method comprises the following steps: the phenotypic sex is male
Example 3
Kit for identifying genetic sex of scatophagus argus
A kit for identifying the genetic sex of a scatophagus argus comprises a primer for identifying the genetic sex of the scatophagus argus; the primer sequences are shown as SEQ ID NO.3 and SEQ ID NO. 4.
Meanwhile, the kit also comprises reagents required by PCR amplification reaction: 2 XPCR mix Buffer and ddH2O。
The use method of the kit comprises the following steps:
s1, extracting genome DNA of a sample to be detected;
s2, carrying out PCR amplification on the genome DNA of the S1 by using the primers to obtain a PCR amplification product;
s3, carrying out electrophoresis on the PCR amplification product of S2, and according to the electrophoresis result, if the sample shows a single band in S2, determining that the fish is female fish; if two clear bands are displayed, the fish is male.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Sequence listing
<110> Guangdong ocean university
<120> specific molecular marker for identifying genetic sex of scatophagus argus and primer and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 593
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tcagagcaca attgttaggc aaagtgaact tctttttgaa tacccagaag ataaaatcac 60
ccaaaattaa ccctttctgg cttacaacaa ataggacaaa tgcagttttc acacttctgt 120
taacttctgc ttctgttaac agtgaatggg cagtgattat ttagatacag tgaactttct 180
tttaatcact catctcagcc aggtgagaag tttactggta gaggttaaca aatcagagac 240
tttcagggag tgttgatctg catataattt caacagtcgg ttttaaagta taattaggac 300
catagactgt ataataattg tggactacca tttgaagcct cgagtgtggc attatggctg 360
tcgctgtctt gggtttgttg agcagaagtg accatatttg gacaagagag ttgagctggg 420
gaggatacgc attgctatgc ctcactcctg actggatctg attgagaacc tgaggacatg 480
ccatggtagt ctcttgtcaa tcagaggttg ccacacctta aagcataccc agcttcatta 540
tctggaccat aatttactca atgaacatca tgctgtattg gtgaaagtag cga 593
<210> 2
<211> 693
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcagagcaca attgttaggc aaagtgaact tctttttgaa tgcccagaag ataaaataac 60
ccaaaataca aaataatgat attataaccc tttctggctt acaacaaata ggactaatgt 120
agttttcaaa cagtatcagg gatgtgcttc tgttaacagt gaatgggcag tgaatgatta 180
tttagataca gtgaactttc ttttaatcag aatatataca gaaatcactg ctgctgtaca 240
tttcaagcac ttggcaaaag tttttcttta atacagtcat ttaaatgtag agtgacatgc 300
aattctttgt gtagtattca tatttgtttt tagtattagt ttgtcagcct tttcaaatct 360
gttttggtcc agtgaactaa taaacaaagt tgatcccaac ctggcctagc aggttgggat 420
cactggtaga ggttaacaaa tcagagaatg atgatctgca tataatttca acactcgatt 480
ttaaagtata cttaggacca tagactgtat aataattgtg gactaccatt tgaagcctcg 540
agtgtggcat tgtggatctg attgagaacc tgaggacatg ccatggtagt ctcttgtcaa 600
tcggaagttg gcacacctta aagcattccc agcttcattg tctggaccat aatttactca 660
atgaacatca tgctgtattg gtgaaagtag cga 693
<210> 3
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcagagcaca attgttaggc aaagtgaac 29
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcgctacttt caccaataca gcatga 26
Claims (10)
1. A specific molecular marker for identifying the genetic sex of the scatophagus argus is characterized in that the molecular marker is two DNA fragments which are partially homologous in an X chromosome and a Y chromosome of the scatophagus argus, and the nucleotide sequences of the DNA fragments which are partially homologous on the X chromosome are shown as SEQ ID NO: 1, the nucleotide sequence of the DNA fragment with partial homology on the Y chromosome is shown as SEQ ID NO: 2, respectively.
2. Use of the specific molecular marker of claim 1 in the preparation of primers for identifying sex of scatophagus argus.
3. A pair of primers capable of PCR amplifying a specific molecular marker or a partial fragment thereof according to claim 1, wherein the PCR amplification product has a significant difference between the X chromosome and the Y chromosome.
4. The primer of claim 3, wherein the nucleotide sequence of the forward primer in the primer is as shown in SEQ ID NO: 3, the nucleotide sequence of the reverse primer is shown as SEQ ID NO: 4, respectively.
5. Use of the specific molecular marker of claim 1 or the primer of claim 3 for identifying the genetic sex of a scatophagus argus.
6. Use of the specific molecular marker of claim 1 or the primer of claim 3 in the preparation of a kit for sex determination of scatophagus argus.
7. A method for rapidly identifying the genetic sex of a scatophagus argus is characterized by comprising the following steps:
s1, extracting genome DNA of a sample to be detected;
s2, designing a primer, and carrying out PCR amplification on the specific molecular marker or a partial fragment thereof according to claim 1 by using the genomic DNA of S1 as a template to obtain a PCR amplification product;
s3, carrying out agarose gel electrophoresis on the PCR amplification product of S2;
s4, analyzing a sequencing result, and judging the genetic sex of the scatophagus argus according to an electrophoresis result, wherein the judgment standard is as follows:
if the PCR amplification product only has a clear band, the sample to be detected is a female fish;
and if the PCR amplification product has two clear bands, the sample to be detected is the male fish.
8. The method of claim 7, wherein the PCR amplification system comprises 2 XPCR mix 25 μ L, 10 μmol/L forward and reverse primers each 1 μ L, genomic DNA2 μ L, ddH2O21. mu.L, 50. mu.L total.
9. The method of claim 7, wherein the PCR amplification procedure comprises: 3min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃ and 1min at 72 ℃ for 37 cycles; extension was carried out at 72 ℃ for 10 min.
10. A kit for identifying the genetic sex of a scatophagus argus, which comprises the primer according to claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010986812.3A CN112029843B (en) | 2020-09-18 | 2020-09-18 | Specific molecular marker for identifying genetic sex of lysimachia christinae as well as primer and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010986812.3A CN112029843B (en) | 2020-09-18 | 2020-09-18 | Specific molecular marker for identifying genetic sex of lysimachia christinae as well as primer and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112029843A true CN112029843A (en) | 2020-12-04 |
| CN112029843B CN112029843B (en) | 2023-07-21 |
Family
ID=73574725
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010986812.3A Active CN112029843B (en) | 2020-09-18 | 2020-09-18 | Specific molecular marker for identifying genetic sex of lysimachia christinae as well as primer and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112029843B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113718043A (en) * | 2021-09-03 | 2021-11-30 | 广东海洋大学 | Specific molecular marker and primer for identifying genetic sex of plectropomus multiformis, and method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009080864A1 (en) * | 2007-12-20 | 2009-07-02 | Riista- Ja Kalatalouden Tutkimuslaitos | Method for the production of fish progeny |
| US20100037330A1 (en) * | 2008-06-11 | 2010-02-11 | Kannika Siripattarapravat | Efficient Somatic Cell Nuclear Transfer In Fish |
| CN107326077A (en) * | 2017-07-14 | 2017-11-07 | 集美大学 | A kind of molecular labeling for differentiating spotted maigre genetic sex and its application |
| CN108192963A (en) * | 2018-03-14 | 2018-06-22 | 广东海洋大学 | A kind of specific molecular marker and its primer of precise Identification Scatophagus argus (Linnaeus) genetic sex |
| KR102018798B1 (en) * | 2018-09-21 | 2019-09-05 | 부경대학교 산학협력단 | The primer set for identification of fish and methods for their qualitative and quantitative analysis |
| CN111000987A (en) * | 2019-12-18 | 2020-04-14 | 广东海洋大学 | Hormone kit for inducing spawning of scatophagus argus and artificial induced spawning and insemination method for obtaining fertilized eggs of scatophagus argus in batches |
-
2020
- 2020-09-18 CN CN202010986812.3A patent/CN112029843B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009080864A1 (en) * | 2007-12-20 | 2009-07-02 | Riista- Ja Kalatalouden Tutkimuslaitos | Method for the production of fish progeny |
| US20100037330A1 (en) * | 2008-06-11 | 2010-02-11 | Kannika Siripattarapravat | Efficient Somatic Cell Nuclear Transfer In Fish |
| CN107326077A (en) * | 2017-07-14 | 2017-11-07 | 集美大学 | A kind of molecular labeling for differentiating spotted maigre genetic sex and its application |
| CN108192963A (en) * | 2018-03-14 | 2018-06-22 | 广东海洋大学 | A kind of specific molecular marker and its primer of precise Identification Scatophagus argus (Linnaeus) genetic sex |
| KR102018798B1 (en) * | 2018-09-21 | 2019-09-05 | 부경대학교 산학협력단 | The primer set for identification of fish and methods for their qualitative and quantitative analysis |
| CN111000987A (en) * | 2019-12-18 | 2020-04-14 | 广东海洋大学 | Hormone kit for inducing spawning of scatophagus argus and artificial induced spawning and insemination method for obtaining fertilized eggs of scatophagus argus in batches |
Non-Patent Citations (14)
| Title |
|---|
| DONG-NENG等: "Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus)", 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY》 * |
| DONG-NENG等: "Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus)", 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY》, vol. 233, 31 December 2019 (2019-12-31), pages 35 * |
| FEI-XIANG HE等: "Comparative transcriptome analysis of male and female gonads reveals sex-biased genes in spotted scat (Scatophagus argus)", 《FISH PHYSIOL BIOCHEM》 * |
| FEI-XIANG HE等: "Comparative transcriptome analysis of male and female gonads reveals sex-biased genes in spotted scat (Scatophagus argus)", 《FISH PHYSIOL BIOCHEM》, vol. 45, no. 6, 31 December 2019 (2019-12-31), pages 1963 - 1980 * |
| UMAR FAROUK MUSTAPHA等: "Male-specific Dmrt1 is a candidate sex determination gene in spotted scat (Scatophagus argus)", 《AQUACULTURE》 * |
| UMAR FAROUK MUSTAPHA等: "Male-specific Dmrt1 is a candidate sex determination gene in spotted scat (Scatophagus argus)", 《AQUACULTURE》, vol. 495, 20 February 2020 (2020-02-20), pages 350 * |
| YUANQING等: "Genome Survey of Male and Female Spotted Scat (Scatophagus argus)", 《ANIMALS (BASEL)》 * |
| YUANQING等: "Genome Survey of Male and Female Spotted Scat (Scatophagus argus)", 《ANIMALS (BASEL)》, vol. 9, no. 12, 11 December 2019 (2019-12-11), pages 1 * |
| 古皓天等: "金钱鱼Dmrt5基因的克隆及表达分析", 《海南热带海洋学院学报》 * |
| 古皓天等: "金钱鱼Dmrt5基因的克隆及表达分析", 《海南热带海洋学院学报》, vol. 26, no. 02, 26 April 2019 (2019-04-26), pages 9 - 15 * |
| 吴波等: "金钱鱼雌雄个体的形态差异分析", 《上海海洋大学学报》 * |
| 吴波等: "金钱鱼雌雄个体的形态差异分析", 《上海海洋大学学报》, vol. 23, no. 01, 15 January 2014 (2014-01-15), pages 64 - 69 * |
| 王耀嵘等: "金钱鱼基因组微卫星分布特征分析及多态性标记开发", 《广东海洋大学学报》 * |
| 王耀嵘等: "金钱鱼基因组微卫星分布特征分析及多态性标记开发", 《广东海洋大学学报》, vol. 40, no. 04, 31 August 2020 (2020-08-31), pages 7 - 14 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113718043A (en) * | 2021-09-03 | 2021-11-30 | 广东海洋大学 | Specific molecular marker and primer for identifying genetic sex of plectropomus multiformis, and method and application thereof |
| CN113718043B (en) * | 2021-09-03 | 2024-03-05 | 广东海洋大学 | Specific molecular marker and primer for identifying genetic sex of Chaptera multiflora and method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112029843B (en) | 2023-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114107522B (en) | Specific marker of female and male genetic sex of oplegnathus punctatus, identification method and kit | |
| CN108192963B (en) | Specific molecular marker for accurately identifying genetic sex of scatophagus argus and primer thereof | |
| WO2016180009A1 (en) | Dna probe sequence for determining genetic sexes of litopenaeus vannamei and obtainment method | |
| CN110607359B (en) | Patinopecten yessoensis female specific marker combination and application | |
| CN111394445B (en) | Indel marker for sex identification of channa maculata and application thereof | |
| CN112048014B (en) | Penaeus monodon PmGLUT2 gene and application thereof | |
| CN104073486A (en) | SNP site related to rapid growth of largemouth black bass as well as identification method and application thereof | |
| CN114836543A (en) | Female molecular marker primer of channa maculata, application thereof and method for identifying sex of channa maculata | |
| CN115896298A (en) | Siniperca chuatsi X chromosome molecular marker primer and application thereof | |
| CN110684776A (en) | Penaeus monodon Na +/K +/2 Cl-co-transporter NKCC gene and application thereof | |
| CN112029843B (en) | Specific molecular marker for identifying genetic sex of lysimachia christinae as well as primer and application thereof | |
| CN111996261B (en) | Macrobrachium rosenbergii sex molecular marker primer and application thereof | |
| CN110551808B (en) | Southern catfish sex chromosome specific molecular marker, genetic sex identification method based on molecular marker and parthenocarpic fish production method | |
| He et al. | Mapping sex-determination region and screening DNA markers for genetic sex identification in largemouth bass (Micropterus salmoides) | |
| CN113718043B (en) | Specific molecular marker and primer for identifying genetic sex of Chaptera multiflora and method and application thereof | |
| CN113604587B (en) | Molecular marker T5198 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof | |
| CN115725746A (en) | Snakehead sex specific molecular marker, genetic sex identification method based on molecular marker and application | |
| CN114480601A (en) | Molecular marker, primer, method and application for identifying genetic sex of hippocampus kelloggi | |
| CN112159852B (en) | Specific molecular marker, detection primer and kit for sex chromosomes of nibea albiflora and application of specific molecular marker, detection primer and kit | |
| CN113699224A (en) | Molecular marker C2 for sex identification of Chinese prawn and application thereof | |
| CN113801945A (en) | Molecular marker C768 for identifying ammonia nitrogen tolerance character of portunus trituberculatus and application thereof | |
| CN113005203A (en) | Microsatellite marking method for identifying paternity of scleropages formosus | |
| CN111748639A (en) | Molecular marker for identifying sex of haliotis discus hannai and application thereof | |
| Zhang et al. | Development and applications of chromosome-specific cytogenetic BAC-FISH probes in Larimichthys crocea | |
| CN113699254B (en) | Sex-specific DNA (deoxyribonucleic acid) marker for large trichromatic and Zhaobiao trichromatic koi and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |