CN109439771B - Method for identifying family of hybrid porgy by using microsatellite marker - Google Patents
Method for identifying family of hybrid porgy by using microsatellite marker Download PDFInfo
- Publication number
- CN109439771B CN109439771B CN201811574004.5A CN201811574004A CN109439771B CN 109439771 B CN109439771 B CN 109439771B CN 201811574004 A CN201811574004 A CN 201811574004A CN 109439771 B CN109439771 B CN 109439771B
- Authority
- CN
- China
- Prior art keywords
- primer
- seq
- sequence
- microsatellite
- porgy
- 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.)
- Active
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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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
-
- 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
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for identifying a family of a hybrid porgy by using a microsatellite marker, belonging to the field of molecular markers. Firstly, cutting parent of genuine porgy female fish and black porgy male fish and tail fin samples of filial generation thereof, extracting DNA, and then taking the DNA of the parent and the filial generation as templates and adopting microsatellite marker primers to carry out multiplex PCR amplification; carrying out polyacrylamide gel electrophoresis on the amplification product, analyzing by using BandScan5.0 software, judging the size of allele by combining the length of the PCR product, determining the genotype of an individual, and establishing a genotype data file of the hybridized porgy and the parent according to the analysis result; the family is judged by using Cervus3.0 software. The invention adopts the microsatellite marking technology to distinguish the genotype of male and female parents for hybridization and offspring on 10 microsatellite loci, can accurately and conveniently determine the family source of the filial generation of the hybridized porgy, quickly and efficiently breeds excellent family of the hybridized porgy, and brings great convenience to the genetic breeding work.
Description
Technical Field
The invention relates to a method for identifying a family of a hybrid porgy by using a microsatellite marker, belonging to the technical field of molecular markers.
Background
The black porgy (Acantho paggrusschlegelii) has oblong and flat side, elegant body shape, beautiful color and luster, and delicious meat, and is a rare marine fish. The black porgy has strong stress resistance, wide temperature and salinity adaptation range, long culture period and poor disease resistance. In recent years, because breeding and improvement of breeding fish are not carried out, germ plasm resources begin to decline, diseases become more serious day by day, quality is reduced, economic benefits obviously decline, and stable and continuous development of the black porgy breeding industry is influenced. Pagrosomus major is a rare marine culture fish species, is a warm-warm bottom carnivorous fish in the inshore, has the characteristics of fast growth, convenient temporary culture, high economic value and the like. In recent years, artificial breeding experiments of Pagrosomus major have achieved certain results, and the Pagrosomus major mariculture fish species have been developed in coastal areas in south China. But the genuine porgy has poor stress resistance and low temperature resistance. Therefore, the genuine porgy and the black porgy are expected to be hybridized in a hybridization breeding mode, so that the hybrid porgy generates heterosis, and the defects that parents are not low-temperature resistant and grow slowly are overcome. 2014. In 2015, researchers of the research institute of marine aquaculture in Jiangsu province develop the technical research on the hybridization breeding of red sea bream and black sea bream in Jiangsu province and at the center of the seedlings, and by taking red sea bream female fish and black sea bream male fish as parents and adopting an artificial insemination method, the filial generation is successfully obtained, and the family of the hybridized red bream is established. The filial generation has the characteristics of standard body type, quick growth, easy capture, high unsaturated fatty acid content and the like, and is a subject for experimental cultivation of coastal marine fishes.
In the process of breeding improved fish species, the paternity relationship or pedigree relationship in the population is clarified, and the inbreeding of the population (individuals) can be avoided. Therefore, the identification of the family of the hybrid porgy is very important, and with the material of the family of the hybrid porgy, the heritability calculation of the growth, stress resistance, fatty acid content and other biological characteristics of the porgy can be carried out, and then the breeding values of various characters are estimated so as to guide the cultivation of new varieties of seawater porgy. However, the aquatic animals are difficult to identify and identify from external characteristics, and the traditional identification mode is difficult to play a role.
Microsatellite DNA (Microsatete DNA), also called Simple Sequence Repeats (SSR) or Short Tandem Repeat (STR), is a Tandem repeat composed of 1-6 bp Short nucleotides as basic units, connected end to end, and composed of a core Sequence and flanking sequences at both sides. Microsatellite DNA is widely distributed in eukaryotic genome, and has the characteristics of high polymorphism, good repeatability, easy detection, codominance and the like, so the microsatellite DNA is widely applied to the researches of genetic structure analysis, genetic relationship identification, genetic linkage map construction and the like of marine organisms. The length of the microsatellite DNA is generally between 100 and 300bp, the microsatellite DNA has low requirements on the quality of a sample during PCR amplification, a partially degraded genome DNA sample can be successfully amplified, the requirement on the sample is low, a very small amount of sample can be obtained by a non-destructive method, and the microsatellite DNA is particularly suitable for the research of endangered species.
By utilizing the characteristics of high polymorphism, high stability and the like of the microsatellite loci, the genetic relationship identification is carried out on the basis of the frequency of each allele of a plurality of loci in a certain population, the identification method has high identification rate, can be used for identifying the blood relationship and the source attribute of a certain individual in a mixed family under a common environment, and brings great convenience for genetic breeding work. So far, there is no method for identifying the family of the hybrid porgy.
Disclosure of Invention
In order to select and breed excellent hybrid porgy families, the invention provides a method for identifying the hybrid porgy families by using microsatellite markers, the identification success rate of the families reaches 99.99 percent, and the method has the characteristics of no harm to experimental animals, convenience, rapidness and high accuracy.
Technical scheme
A method for identifying a family of a hybrid porgy by using a microsatellite marker comprises the following steps:
(1) shearing parent of red sea bream and tail fin sample of filial generation thereof, and extracting genome DNA;
(2) taking DNA of the parents and filial generations in the step (1) as templates, and adopting a microsatellite marker primer to carry out multiple PCR amplification;
(3) performing polyacrylamide gel electrophoresis on the PCR amplification product obtained in the step (2), performing silver staining and color development, analyzing an electrophoresis pattern by using BandScan5.0 software, judging the size of an allele by combining the length of the PCR product, and determining the genotype of an individual;
(4) establishing genotype data files of the hybrid porgy and the parents thereof according to the analysis result of the step (3);
(5) the family is judged by using Cervus3.0 software.
In the step (1), the extraction method of the genome DNA of the parent and the filial generation of the female red sea bream and the male black sea bream comprises the following steps:
1) taking 5-20 mg of fin tissue, cutting into pieces, placing the pieces into a 1.5mL centrifuge tube, adding 400 mu L of tissue extracting solution and 10 mu L of proteinase K, shaking and uniformly mixing on an oscillator for 1min, and then placing the mixture in a 55 ℃ constant-temperature water bath kettle for water bath digestion for 2.5 h;
2) adding 300 mu L of Ext solution and 300 mu L of LAB solution into a sample, uniformly mixing, centrifuging at 12000rpm for 5min, taking a lower-layer water phase, placing in a GenClean column, centrifuging at 8000rpm for 1min, and removing waste liquid;
3) adding 500 μ L eluent into GenClean column, and centrifuging at 8000rpm for 1 min;
4) repeating the step 3) once;
5) taking out the GenClean column, discarding the waste liquid in the collecting pipe, putting the GenClean column back into the collecting pipe, and centrifuging at 12000rpm at room temperature for 1min to remove the residual eluent;
6) putting a GenClean column into a new clean 1.5ml centrifuge tube, adding 50-100 μ L of precipitation Buffer in the center of the column, standing at room temperature or 55 ℃ for 2min, centrifuging at 12000rpm for 1min, taking the liquid in the centrifuge tube as genome DNA, and finally storing at-20 ℃ for later use.
Further, in the step (2), the microsatellite marker primers are 10 groups, the upstream primer sequence of the microsatellite marker primer 1 is shown AS SEQ ID No.1, the downstream primer sequence is shown AS SEQ ID No.2, and the marker locus is AS 1;
the sequence of the upstream primer of the microsatellite marker primer 2 is shown AS SEQ ID NO.3, the sequence of the downstream primer is shown AS SEQ ID NO.4, and the marker locus is AS 2;
the sequence of the upstream primer of the microsatellite marker primer 3 is shown AS SEQ ID NO.5, the sequence of the downstream primer is shown AS SEQ ID NO.6, and the marker locus is AS 3;
the sequence of the upstream primer of the microsatellite marker primer 4 is shown AS SEQ ID NO.7, the sequence of the downstream primer is shown AS SEQ ID NO.8, and the marker locus is AS 4;
the sequence of the upstream primer of the microsatellite marker primer 5 is shown AS SEQ ID NO.9, the sequence of the downstream primer is shown AS SEQ ID NO.10, and the marker locus is AS 5;
the sequence of the upstream primer of the microsatellite marker primer 6 is shown AS SEQ ID NO.11, the sequence of the downstream primer is shown AS SEQ ID NO.12, and the marker locus is AS 6;
the sequence of the upstream primer of the microsatellite marker primer 7 is shown AS SEQ ID NO.13, the sequence of the downstream primer is shown AS SEQ ID NO.14, and the marker locus is AS 7;
the sequence of the upstream primer of the microsatellite marker primer 8 is shown AS SEQ ID NO.15, the sequence of the downstream primer is shown AS SEQ ID NO.16, and the marker locus is AS 8;
the sequence of the upstream primer of the microsatellite marker primer 9 is shown AS SEQ ID NO.17, the sequence of the downstream primer is shown AS SEQ ID NO.18, and the marker locus is AS 9;
the sequence of the upstream primer of the microsatellite marker primer 10 is shown AS SEQ ID NO.19, the sequence of the downstream primer is shown AS SEQ ID NO.20, and the marker locus is AS 10.
Further, in the step (2), the reaction system (25 μ L) for PCR amplification was: ddH2O17.25. mu.L, 10 XBuffer 2.5. mu.L, dNTP 0.5. mu.L, primer 2. mu.L, genomic DNA 1. mu.L, Mg2+1.5 μ L, Taq enzyme 0.25 μ L.
Further, in the step (2), the reaction conditions for PCR amplification are as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 40s, and annealing at 57.8-62.0 ℃ for 40 s; extension at 72 ℃ for 1min for 30 cycles; finally, extension is carried out for 10min at 72 ℃.
Further, in the step (3), 8% non-denaturing polyacrylamide gel is adopted for polyacrylamide gel electrophoresis.
The invention has the beneficial effects that: the invention adopts the microsatellite marking technology, distinguishes the genotypes of male and female parents and offspring for hybridization on 10 microsatellite loci, can accurately and conveniently determine the family source of the filial generation of the hybridized porgy, clearly records the family spectrum of the filial generation, determines the genotype of the hybridized porgy aiming at the excellent characters of quick growth, strong stress resistance and the like reflected in production, selects the excellent individual genotype in the breeding process, eliminates the poor individual genotype, can more quickly and efficiently breed the excellent family of the hybridized porgy, brings great convenience for genetic breeding work, and realizes the goal of molecular marker-assisted breeding.
Drawings
FIG. 1 is an electrophoretogram of AS4 at the site in parent and hybrid porgy populations;
FIG. 2 is an electrophoretogram of AS8 at the site in the parent and hybrid porgy populations.
Detailed Description
The technical solution of the present invention is further explained with reference to the accompanying drawings and specific embodiments.
Example 1
Selecting 10 red sea breams (marked as P) with mature gonads from 200 red sea breams Fujian population in 4 months in 2014R1-PR10) And selecting 12 male black porgy fish with mature gonad from 260 black porgy Jiangsu population (marked as P)B1-PB12) Matching according to a double-row hybridization mode, establishing 120 families by an artificial insemination method, cutting tail fin rays when the hybridized individuals grow for 90 days, storing in 100% alcohol, and carrying out genetic relationship identification on 20 hybridized sea bream filial generation individuals (marked as HF1-HF 20).
A method for identifying a family of a hybrid porgy by using a microsatellite marker comprises the following steps:
(1) shearing parent of red sea bream and tail fin sample of filial generation thereof, and extracting genome DNA;
1) taking 5-20 mg of fin tissue, cutting into pieces, placing the pieces into a 1.5mL centrifuge tube, adding 400 mu L of tissue extracting solution and 10 mu L of proteinase K, shaking and uniformly mixing on an oscillator for 1min, and then placing the mixture in a 55 ℃ constant-temperature water bath kettle for water bath digestion for 2.5 h;
2) adding 300 mu L of Ext solution and 300 mu L of LAB solution into a sample, uniformly mixing, centrifuging at 12000rpm for 5min, taking a lower-layer water phase, placing in a GenClean column, centrifuging at 8000rpm for 1min, and removing waste liquid;
3) adding 500 μ L eluent into GenClean column, and centrifuging at 8000rpm for 1 min;
4) repeating the step 3) once;
5) taking out the GenClean column, discarding the waste liquid in the collecting pipe, putting the GenClean column back into the collecting pipe, and centrifuging at 12000rpm at room temperature for 1min to remove the residual eluent;
6) putting a GenClean column into a new clean 1.5ml centrifuge tube, adding 50-100 μ L of precipitation Buffer in the center of the column, standing at room temperature or 55 ℃ for 2min, centrifuging at 12000rpm for 1min, taking the liquid in the centrifuge tube as genome DNA, and finally storing at-20 ℃ for later use.
(2) Taking the DNA of the parents and 20 filial generations in the step (1) as a template, and adopting a microsatellite marker primer to carry out multiple PCR amplification; the microsatellite marker primers and annealing temperatures are shown in Table 1:
TABLE 1
The reaction system for PCR amplification (25. mu.L) was: ddH2O17.25. mu.L, 10 XBuffer 2.5. mu.L, dNTP 0.5. mu.L, primer 2. mu.L, genomic DNA 1. mu.L, Mg2+1.5 μ L, Taq enzyme 0.25 μ L.
The reaction conditions for PCR amplification are as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 40s and annealing for 40 s; extension at 72 ℃ for 1min for 30 cycles; finally, extension is carried out for 10min at 72 ℃.
(3) Performing polyacrylamide gel electrophoresis on the PCR amplification product obtained in the step (2), performing silver staining and color development, analyzing an electrophoresis pattern by using BandScan5.0 software, judging the size of an allele by combining the length of the PCR product, and determining the genotype of an individual;
(4) establishing genotype data files of the hybrid porgy and the parents thereof according to the analysis result of the step (3);
(5) the family is judged by using Cervus3.0 software.
FIG. 1 is an electrophoretogram of site AS4 on parent and hybrid Pagrus major, wherein the electrophoretogram has 43 tracks, and from left to right, HF1, HF2, HF3, HF4, HF5, HF6, HF7, HF8, HF9, HF10, HF11, HF12, HF13, HF14, HF15, HF16, HF17, HF18, HF19, HF20, M, PR1、PR2、PR3、PR4、PR5、PR6、PR7、PR8、PR9、PR10、PB1、PB2、PB3、PB4、PB5、PB6、PB7、PB8、PB9、PB10、PB11、PB12, wherein PRIndicating the female Pagrosomus, PBIndicates the male fish of black porgy, HF indicates the filial generation, M indicates Marker, as shown in FIG. 1It is seen that: at the microsatellite AS4 site, P in female Pagrosomus major parentR1、PR3、PR5、PR7、PR8 is a homozygote, PR2、PR4、PR6、PR9、PR10 is a hybrid, P in male black porgy parentB3、PB4、PB6、PB7、PB9、PB10 is a homozygote, PB1、PB2、PB5、PB8、PB11、PB12 is a hybrid, and individuals HF10, HF15, HF18, and HF20 are homozygotes among offspring, and others are heterozygotes.
FIG. 2 is an electrophoretogram of the parent and hybrid Pagrus major at the site AS8, wherein the electrophoretogram has 43 tracks and P sequentially from left to rightR1、PR2、PR3、PR4、PR5、PR6、PR7、PR8、PR9、PR10、PB1、PB2、PB3、PB4、PB5、PB6、PB7、PB8、PB9、PB10、PB11、PB12. M, HF1, HF2, HF3, HF4, HF5, HF6, HF7, HF8, HF9, HF10, HF11, HF12, HF13, HF14, HF15, HF16, HF17, HF18, HF19, HF20, wherein P is a metal oxide, and P is a metal oxideRIndicating the female Pagrosomus, PBIndicates a black porgy male fish, HF indicates a filial generation, M indicates Marker, as can be seen from fig. 2: at the microsatellite AS8 site, P in female Pagrosomus major parentR2、PR4、PR5、PR6、PR8、PR10 is a homozygote, PR1、PR3、PR7、PR9 is a hybrid, P in male black porgy parentB3、PB5、PB8、PB10、PB11、PB12 is a homozygote, PB1、PB2、PB4、PB6、PB7、PB9 is a heterozygote, and only HF4 is a homozygote in the offspring, and the others are heterozygotes.
After determining the genotype of each locus, a database can be built. All progeny genotype data from this example were analyzed using cervus3.0 software to calculate allele frequencies and genetic parameters, see table 2:
TABLE 2
Remarking: na, allelic factor; n is a radical ofeEffective allelic factor; ho, observing heterozygosity; he, desired heterozygosity; PIC, polymorphic information content.
As can be seen from Table 2, the number of alleles of the selected microsatellite loci is more than 3, the content of polymorphic information is 0.2695-0.8823, and the selected microsatellite loci have more alleles and rich polymorphic information content and are suitable for paternity test and germplasm genetic diversity analysis.
7 sites can be effectively amplified in the hybridization porgy group, and the genetic parameters are all larger than those of the black porgy group, which indicates that the hybridization offspring has higher genetic diversity than the black porgy.
After the genotype data file is established, the parents of 20 foreign porgy are identified by using the parents with known sex, and the result is shown in table 3:
TABLE 3
It can be seen that 20 individuals were successfully classified into different families, wherein 6 individuals (HF1, HF3, HF4, HF7, HF9, HF11) (denoted as family 1) were from a pair of parent parents (PR1, PB1), 4 individuals (HF2, HF5, HF8, HF10) (denoted as family 2) were from another pair of parent parents (PR3, PB4), 2 individuals (HF14, HF16) shared parent 3 with family 2, 2 individuals (HF15, HF18) shared parent PB1 with family 1, and the rest were from other parent parents, respectively.
Sequence listing
<110> research institute of marine aquaculture in Jiangsu province
<120> a method for identifying a family of a hybrid porgy using microsatellite markers
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 1
<210> 2
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 2
<210> 3
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 3
<210> 4
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 4
<210> 5
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 5
<210> 6
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 6
<210> 7
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 7
<210> 8
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 8
<210> 9
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 9
<210> 10
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 10
<210> 11
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 11
<210> 12
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 12
<210> 13
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 13
<210> 14
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 14
<210> 15
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 15
<210> 16
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 16
<210> 17
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 17
<210> 18
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 18
<210> 19
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 19
<210> 20
<211> 3
<212> DNA
<213> microsatellite DNA (Microsatelite DNA)
<400> 20
Claims (4)
1. A method for identifying a family of a hybrid porgy by using a microsatellite marker is characterized by comprising the following steps:
(1) shearing parent of red sea bream and tail fin sample of filial generation thereof, and extracting genome DNA;
(2) taking DNA of the parents and filial generations in the step (1) as templates, and adopting a microsatellite marker primer to carry out multiple PCR amplification;
(3) performing polyacrylamide gel electrophoresis on the PCR amplification product obtained in the step (2), performing silver staining and color development, analyzing an electrophoresis pattern by using BandScan5.0 software, judging the size of an allele by combining the length of the PCR product, and determining the genotype of an individual;
(4) establishing genotype data files of the hybrid porgy and the parents thereof according to the analysis result of the step (3);
(5) family judgment is carried out by using Cervus3.0 software;
in the step (2), the microsatellite marker primers comprise 10 groups, the upstream primer sequence of the microsatellite marker primer 1 is shown AS SEQ ID NO.1, the downstream primer sequence is shown AS SEQ ID NO.2, and the marker locus is AS 1;
the sequence of the upstream primer of the microsatellite marker primer 2 is shown AS SEQ ID NO.3, the sequence of the downstream primer is shown AS SEQ ID NO.4, and the marker locus is AS 2;
the sequence of the upstream primer of the microsatellite marker primer 3 is shown AS SEQ ID NO.5, the sequence of the downstream primer is shown AS SEQ ID NO.6, and the marker locus is AS 3;
the sequence of the upstream primer of the microsatellite marker primer 4 is shown AS SEQ ID NO.7, the sequence of the downstream primer is shown AS SEQ ID NO.8, and the marker locus is AS 4;
the sequence of the upstream primer of the microsatellite marker primer 5 is shown AS SEQ ID NO.9, the sequence of the downstream primer is shown AS SEQ ID NO.10, and the marker locus is AS 5;
the sequence of the upstream primer of the microsatellite marker primer 6 is shown AS SEQ ID NO.11, the sequence of the downstream primer is shown AS SEQ ID NO.12, and the marker locus is AS 6;
the sequence of the upstream primer of the microsatellite marker primer 7 is shown AS SEQ ID NO.13, the sequence of the downstream primer is shown AS SEQ ID NO.14, and the marker locus is AS 7;
the sequence of the upstream primer of the microsatellite marker primer 8 is shown AS SEQ ID NO.15, the sequence of the downstream primer is shown AS SEQ ID NO.16, and the marker locus is AS 8;
the sequence of the upstream primer of the microsatellite marker primer 9 is shown AS SEQ ID NO.17, the sequence of the downstream primer is shown AS SEQ ID NO.18, and the marker locus is AS 9;
the sequence of the upstream primer of the microsatellite marker primer 10 is shown AS SEQ ID NO.19, the sequence of the downstream primer is shown AS SEQ ID NO.20, and the marker locus is AS 10.
2. The method for identifying a family of hybrid Pagrus major using microsatellite markers as set forth in claim 1Characterized in that in the step (2), the reaction system (25 muL) for PCR amplification is as follows: ddH2O17.25. mu.L, 10 XBuffer 2.5. mu.L, dNTP 0.5. mu.L, primer 2. mu.L, genomic DNA 1. mu.L, Mg2+1.5 μ L, Taq enzyme 0.25 μ L.
3. The method for identifying a family of hybrid porgy using microsatellite markers as set forth in claim 1, wherein in the step (2), the PCR amplification is carried out under the following conditions: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 40s, and annealing at 57.8-62.0 ℃ for 40 s; extension at 72 ℃ for 1min for 30 cycles; finally, extension is carried out for 10min at 72 ℃.
4. The method for identifying a family of hybrid porgy using microsatellite markers according to claim 1, 2 or 3 wherein in step (3) said polyacrylamide gel electrophoresis is carried out using 8% native polyacrylamide gel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811574004.5A CN109439771B (en) | 2018-12-21 | 2018-12-21 | Method for identifying family of hybrid porgy by using microsatellite marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811574004.5A CN109439771B (en) | 2018-12-21 | 2018-12-21 | Method for identifying family of hybrid porgy by using microsatellite marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109439771A CN109439771A (en) | 2019-03-08 |
| CN109439771B true CN109439771B (en) | 2021-07-02 |
Family
ID=65534749
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811574004.5A Active CN109439771B (en) | 2018-12-21 | 2018-12-21 | Method for identifying family of hybrid porgy by using microsatellite marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109439771B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667760B (en) * | 2021-07-06 | 2023-07-14 | 中山大学 | SSR (simple sequence repeat) marker primer and method for evaluating genetic diversity of sparus praecox population |
| CN114438221A (en) * | 2022-01-11 | 2022-05-06 | 江苏省海洋水产研究所 | SNP molecular marker related to black porgy growth traits and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611405B (en) * | 2018-04-25 | 2022-02-15 | 浙江省淡水水产研究所 | Microsatellite fluorescent multiplex PCR (polymerase chain reaction) method for parent-child identification of culter alburnus |
| CN108588232B (en) * | 2018-04-28 | 2022-02-15 | 中国水产科学研究院淡水渔业研究中心 | Mandarin fish paternity testing kit and microsatellite PCR (polymerase chain reaction) identification method thereof |
-
2018
- 2018-12-21 CN CN201811574004.5A patent/CN109439771B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109439771A (en) | 2019-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107164463B (en) | SNP marker for determining and/or genetically improving growth traits of pigs | |
| CN109439771B (en) | Method for identifying family of hybrid porgy by using microsatellite marker | |
| CN116356038A (en) | Breeding method for screening Fugu rubripes individuals with rapid growth performance | |
| CN109825603B (en) | SNP molecular marker related to growth traits of hybrid pelteobagrus fulvidraco 'Huangyou No. 1' and application thereof | |
| CN109182557B (en) | SNP molecular marker for identifying low dissolved oxygen tolerance and fullness of pelteobagrus vachelli and application thereof | |
| CN111979336B (en) | Specific primer and method for identifying fugu obscurus, fugu rubripes and hybrid fugu rubripes | |
| CN107475413B (en) | Method for screening crassostrea gigas parent shellfish with high content of unsaturated fatty acid C20:3 omega 6 | |
| CN111073983B (en) | SNP marker related to identification of northern subspecies and Florida subspecies of largemouth bass and application thereof | |
| CN111057771B (en) | SNP molecular marker for distinguishing 'Zhongyang No. 1' from common fugu obscurus and application thereof | |
| CN113502336B (en) | Siniperca chuatsi hypoxia-resistant character-related SNP molecular marker and application thereof | |
| CN113604587B (en) | Molecular marker T5198 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof | |
| CN113249497B (en) | SNP molecular marker related to growth traits of mandarin fish, primer and application | |
| CN113736891B (en) | Molecular marker G2997 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof | |
| Shen et al. | Construction of genetic linkage maps of guppy (Poecilia reticulata) based on AFLP and microsatellite DNA markers | |
| CN111378765B (en) | SNP (Single nucleotide polymorphism) marker of fast-growing grass carp individual and application of SNP marker | |
| CN111793699B (en) | Efficient matching and breeding method for procypris merus | |
| CN108841930B (en) | Paramisgurnus dabryanus microsatellite family identification method and application thereof | |
| CN113584188A (en) | Low-temperature-resistant molecular marker C6101 of penaeus japonicus and application | |
| CN113684285A (en) | Trachinotus ovatus cryptocaryon irritans disease associated SNP molecular marker, primer and application thereof | |
| CN113584187A (en) | Molecular marker A2629 for screening penaeus japonicus with low temperature resistance, amplification primer and application thereof | |
| CN117487931B (en) | Sillago sihama hypoxia tolerance character related SNP molecular marker and application thereof | |
| CN113373142B (en) | Molecular marker-assisted selection method for pig backfat thickness and application thereof | |
| CN112322756B (en) | SNP locus linked with growth trait of fugu rubripes | |
| CN110724750B (en) | Molecular marker C242 of portunus trituberculatus parahaemolyticus and application thereof | |
| CN113621714B (en) | Low-temperature-resistant molecular marker A257 of penaeus japonicus 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 |