CN109439771A - A method of hybridization porgy family is identified using microsatellite marker - Google Patents
A method of hybridization porgy family is identified using microsatellite marker Download PDFInfo
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- 238000011144 upstream manufacturing Methods 0.000 claims description 30
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Abstract
The present invention relates to a kind of methods for identifying hybridization porgy family using microsatellite marker, belong to field of molecular marker.Clip red porgy raun and black porgy milter parent and its filial generation tail fin sample first, carry out the extraction of DNA, then using parent and the DNA of filial generation as template, carry out multiplexed PCR amplification using microsatellite marker primer；Polyacrylamide gel electrophoresis is carried out to amplified production, is analyzed with BandScan5.0 software, is judged allele size in conjunction with PCR product length, determine idiotype, establishes hybridization porgy and the genotype data file of parent based on the analysis results；Family differentiation is carried out using Cervus3.0 software.The present invention uses micro-satellite labeling technique, hybridization female male parent and offspring's genotype are distinguished on 10 microsatellite seats, the family source that can accurately, easily determine hybridization porgy offspring, quickly and efficiently selects excellent hybridization porgy family, brings great convenience for genetic breeding work.
Description
Technical field
The present invention relates to a kind of methods for identifying hybridization porgy family using microsatellite marker, belong to molecular marking technique neck
Domain.
Background technique
Black porgy (Acantho pagrusschlegelii) bodily form is oblong, flat-sided, posture is graceful, color element is beautiful,
Delicious meat is a kind of rare seafood fish.The resistance of black porgy fish is strong, wide to temperature, salinity adaptation range, but cultivates week
Phase is long, premunition is poor.In recent years due to not carrying out breeding and improvement to kind of fish, germ plasm resource, which has begun, to fail, disease
Evil is on the rise, quality decline, and economic benefit obviously glides, and affects the stabilization and sustainable development of black porgy aquaculture.Red porgy
(Pagrosomus major) is that warm nature bottom predacious fish is warmed up in coastal waters, have growth it is fast, convenient for temporarily supporting, economic value height etc.
Feature is a kind of rare sea-farming fingerling.In recent years, the artificial breeding test of red porgy has achieved certain achievement,
Coastal areas of southern China area has been developed as a kind of main sea-farming fingerling.But red porgy resistance is not poor, low temperature resistant.Cause
This, it may be desirable that by way of crossbreeding, red porgy is hybridized with black porgy, the advantage so that hybridization porgy hybridizes, gram
Take the disadvantage that parent is not low temperature resistant, growth is partially slow.2014, Jiangsu Prov. Inst. of Marine Aquatic Products scientific research personnel in 2015 is in Jiangsu Province
The crossbreeding technology research of red porgy black porgy has been carried out at marine culture and stock enhancement technology and seedling center, with red porgy raun and black porgy milter
Filial generation is had successfully been obtained using the method for artificial insemination for parent, establishes hybridization porgy family.Filial generation has body
Type standard, fast, the easy harvesting of growth, the features such as unsaturated fatty acid content is high, are the objects of coastal seawater fish test cultivation.
During fish fine-variety breeding, makes the parent child relationship or genealogical relationship in population clear, it is (a to can be avoided population
Body) inbreeding occurs.Therefore, hybridization porgy family identify extremely important, have hybridization porgy man based material, madai can be carried out
The biological characteristics genetic force such as growth, degeneration-resistant, content of fatty acid calculate, and then estimate the breeding value of each character, to refer to
Lead the cultivation of seawater madai new varieties.But aquatic livestock is due to being difficult to identify, being not easy to identify from surface, traditional identification method
It is difficult to play a role.
Microsatellite DNA (Microsatellite DNA), also known as simple repeated sequence (Simple Sequence
Repeats, SSR) or short tandem repeat (Short Tandem Repeats, STR), refer to the short nucleotide with 1~6bp
For basic unit, the tandem repetitive sequence of end to end composition is made of the flanking sequence of core sequence and two sides.Microsatellite
DNA is distributed widely in eukaryotic gene group, and because have polymorphism it is high, it is reproducible, be easy to the spies such as detection, codominance
Property, it is widely used in the researchs such as the genetic structure analyses of marine organisms, Relationship iden- tification, genetic linkage maps building.
The length of microsatellite DNA is generally between 100~300bp, when PCR amplification, Partial digestion not high to the quality requirement of sample
Genome DNA sample also can Successful amplification, and few to the demand of sample, the minute quantity that can be obtained with non-damage method
Sample is particularly suitable for the research of endangered species.
Using the high polymorphism of microsatellite locus, the features such as high stability, with multiple sites in certain group each equipotential
Relationship iden- tification is carried out based on the frequency of gene, there is very high discrimination, be can be used for identifying and be mixed under common environmental
The genetic connection and source attribute of some individual in being of the whole family, bring great convenience for genetic breeding work.So far, also
The identification method of porgy family is not hybridized.
Summary of the invention
For the excellent hybridization porgy family of breeding, microsatellite marker identification hybridization porgy family is utilized the present invention provides a kind of
Method, the identification success rate of the family up to 99.99%, the present invention have not damage experiment animal, convenient and efficient, accuracy rate is high
The characteristics of.
Technical solution
A method of hybridization porgy family is identified using microsatellite marker, is included the following steps:
(1) clip red porgy raun and black porgy milter parent and its filial generation tail fin sample, carry out mentioning for genomic DNA
It takes；
(2) using parent in step (1) and the DNA of filial generation as template, multiplex PCR is carried out using microsatellite marker primer
Amplification；
(3) polyacrylamide gel electrophoresis, silver staining colour developing are carried out to the pcr amplification product of step (2), electrophorogram is used
The analysis of BandScan5.0 software, judges allele size in conjunction with PCR product length, determines idiotype；
(4) hybridization porgy and its genotype data file of parent are established according to the analysis result of step (3)；
(5) family differentiation is carried out using Cervus3.0 software.
In step (1), the extracting method of the genomic DNA of red porgy raun and black porgy milter parent and its filial generation are as follows:
1) fin 5~20mg of tissue is taken, shreds and is placed in 1.5mL centrifuge tube, 400 μ L tissue extracts and 10 μ L are added
Proteinase K, concussion mixes 1min on the oscillator, is then placed within water-bath in 55 DEG C of thermostat water baths and digests 2.5h；
2) 300 μ L Ext solution and 300 μ L AB solution are added into sample, after mixing, 12000rpm from
Heart 5min takes lower layer's water phase to be placed in GenClean column, after 8000rpm is centrifuged 1min, removes waste liquid；
3) 500 μ L eluents are added into GenClean column, 8000rpm is centrifuged 1min；
4) it is primary to repeat step 3)；
5) GenClean column is taken out, the waste liquid in collecting pipe is discarded, GenClean column is put back in collecting pipe,
12000rpm, room temperature is centrifuged 1min, to remove residual eluent；
6) GenClean column is put into new clean 1.5ml centrifuge tube, 50~100 μ L Elution is added in column center
After Buffer, room temperature or 55 DEG C of placement 2min, 12000rpm is centrifuged 1min, and the liquid in centrifuge tube is genomic DNA, finally
- 20 DEG C are placed in save backup.
Further, in step (2), microsatellite marker primer is 10 groups, and the upstream primer sequence of microsatellite marker primer 1 is such as
Shown in SEQ ID NO.1, downstream primer sequence is as shown in SEQ ID NO.2, marker site AS1；
The upstream primer sequence of microsatellite marker primer 2 is as shown in SEQ ID NO.3, downstream primer sequence such as SEQ ID
Shown in NO.4, marker site AS2；
The upstream primer sequence of microsatellite marker primer 3 is as shown in SEQ ID NO.5, downstream primer sequence such as SEQ ID
Shown in NO.6, marker site AS3；
The upstream primer sequence of microsatellite marker primer 4 is as shown in SEQ ID NO.7, downstream primer sequence such as SEQ ID
Shown in NO.8, marker site AS4；
The upstream primer sequence of microsatellite marker primer 5 is as shown in SEQ ID NO.9, downstream primer sequence such as SEQ ID
Shown in NO.10, marker site AS5；
The upstream primer sequence of microsatellite marker primer 6 is as shown in SEQ ID NO.11, downstream primer sequence such as SEQ ID
Shown in NO.12, marker site AS6；
The upstream primer sequence of microsatellite marker primer 7 is as shown in SEQ ID NO.13, downstream primer sequence such as SEQ ID
Shown in NO.14, marker site AS7；
The upstream primer sequence of microsatellite marker primer 8 is as shown in SEQ ID NO.15, downstream primer sequence such as SEQ ID
Shown in NO.16, marker site AS8；
The upstream primer sequence of microsatellite marker primer 9 is as shown in SEQ ID NO.17, downstream primer sequence such as SEQ ID
Shown in NO.18, marker site AS9；
The upstream primer sequence of microsatellite marker primer 1 is as shown in SEQ ID NO.19, downstream primer sequence such as SEQ ID
Shown in NO.20, marker site AS10.
Further, in step (2), the reaction system (25 μ L) of PCR amplification are as follows: ddH2O 17.25 μ L, 10 × Buffer
2.5 μ L, dNTP 0.5 μ L, primer 2 μ L, genomic DNA 1 μ L, Mg2+1.5 μ L, 0.25 μ L of Taq enzyme.
Further, in step (2), the reaction condition of PCR amplification are as follows: 94 DEG C of initial denaturation 10min；94 DEG C of denaturation 40s,
57.8-62.0 DEG C of annealing 40s；72 DEG C of extension 1min, 30 circulations；Last 72 DEG C of extensions 10min.
Further, in step (3), the polyacrylamide gel electrophoresis using 8% non-denaturing polyacrylamide
Gel.
Beneficial effects of the present invention: the present invention uses micro-satellite labeling technique, and hybridization is distinguished on 10 microsatellite seats
With the genotype of female male parent and offspring, it can accurately, easily determine the family source of hybridization porgy offspring, its pedigree is carried out
Specific to record, fast for the upper reflected growth of production, resistance waits by force merits, its genotype is determined, in breeding
In the process, defect individual genotype is selected, bad idiotype is eliminated, can more quickly and efficiently select excellent hybridization
Porgy family brings great convenience for genetic breeding work, realizes the target of molecular mark.
Detailed description of the invention
Fig. 1 is site AS4 in parent and the electrophoretogram of hybridization porgy group；
Fig. 2 is site AS8 in parent and the electrophoretogram of hybridization porgy group.
Specific embodiment
Technical solution of the present invention is described further in the following with reference to the drawings and specific embodiments.
Embodiment 1
In April, 2014, the red porgy raun that 10 tail gonadal maturations are chosen from 200 tail red porgy Fujian groups (are denoted as
PR1-PR10) and the black porgy milter of 12 tail gonadal maturations is selected (to be denoted as P in 260 tail black porgy Jiangsu groupsB1-PB12) it, presses
Diallel cross mode carries out combo, establishes 120 familys by the method for artificial insemination, grows to 90 days to hybrid individual,
Clip tail fin fin ray, is put into the preservation of 100% alcohol, carries out affiliation to 20 hybridization porgy offspring individuals (being denoted as HF1-HF20)
Identification.
A method of hybridization porgy family is identified using microsatellite marker, is included the following steps:
(1) clip red porgy raun and black porgy milter parent and its filial generation tail fin sample, carry out mentioning for genomic DNA
It takes；
1) fin 5~20mg of tissue is taken, shreds and is placed in 1.5mL centrifuge tube, 400 μ L tissue extracts and 10 μ L are added
Proteinase K, concussion mixes 1min on the oscillator, is then placed within water-bath in 55 DEG C of thermostat water baths and digests 2.5h；
2) 300 μ L Ext solution and 300 μ LAB solution are added into sample, after mixing, 12000rpm from
Heart 5min takes lower layer's water phase to be placed in GenClean column, after 8000rpm is centrifuged 1min, removes waste liquid；
3) 500 μ L eluents are added into GenClean column, 8000rpm is centrifuged 1min；
4) it is primary to repeat step 3)；
5) GenClean column is taken out, the waste liquid in collecting pipe is discarded, GenClean column is put back in collecting pipe,
12000rpm, room temperature is centrifuged 1min, to remove residual eluent；
6) GenClean column is put into new clean 1.5ml centrifuge tube, 50~100 μ L Elution is added in column center
After Buffer, room temperature or 55 DEG C of placement 2min, 12000rpm is centrifuged 1min, and the liquid in centrifuge tube is genomic DNA, finally
- 20 DEG C are placed in save backup.
(2) it using parent in step (1) and the DNA of 20 filial generations as template, is carried out using microsatellite marker primer more
Weight PCR amplification；Microsatellite marker primer and annealing temperature are shown in Table 1:
Table 1
The reaction system (25 μ L) of PCR amplification are as follows: ddH217.25 2.5 0.5 μ L of μ L, dNTP of μ L, 10 × Buffer of O,
Primer 2 μ L, genomic DNA 1 μ L, Mg2+1.5 μ L, 0.25 μ L of Taq enzyme.
The reaction condition of PCR amplification are as follows: 94 DEG C of initial denaturation 10min；94 DEG C of denaturation 40s, annealing 40s；72 DEG C of extension 1min,
30 circulations；Last 72 DEG C of extensions 10min.
(3) polyacrylamide gel electrophoresis, silver staining colour developing are carried out to the pcr amplification product of step (2), electrophorogram is used
The analysis of BandScan5.0 software, judges allele size in conjunction with PCR product length, determines idiotype；
(4) hybridization porgy and its genotype data file of parent are established according to the analysis result of step (3)；
(5) family differentiation is carried out using Cervus3.0 software.
Fig. 1 is that site AS4 shares 43 runways on parent and the electrophoretogram of hybridization porgy group, electrophoretogram, from left to right
Be followed successively by 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 PRIndicate red porgy raun, PBIndicate black porgy milter, HF indicates hybridization
Offspring, M indicate Marker, as seen from Figure 1: the P in the site microsatellite AS4, female red porgy parentR1、PR3、PR5、PR7、
PR8 be homozygote, PR2、PR4、PR6、PR9、PR10 be heterozygote, P in male black porgy parentB3、PB4、PB6、PB7、PB9、PB10 are
Homozygote, PB1、PB2、PB5、PB8、PB11、PB12 be heterozygote, and HF10, HF15, HF18 and HF20 individual are homozygosis in offspring
Son, other are all heterozygote.
Fig. 2 is that site AS8 shares 43 runways on parent and the electrophoretogram of hybridization porgy group, electrophoretogram, from left to right
It is followed successively by PR1、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 PRIndicate red porgy raun, PBAfter indicating that black porgy milter, HF indicate hybridization
Generation, M indicate Marker, as seen from Figure 2: the P in the site microsatellite AS8, female red porgy parentR2、PR4、PR5、PR6、PR8、
PR10 be homozygote, PR1、PR3、PR7、PR9 be heterozygote, P in male black porgy parentB3、PB5、PB8、PB10、PB11、PB12 be pure
Zoarium, PB1、PB2、PB4、PB6、PB7、PB9 be heterozygote, in offspring only No. HF4 be homozygote, other are all heterozygote.
After determining each loci gene type, database can be established.Using Cervus3.0 software to filial generations all in the present embodiment
Genotype data is analyzed, and is calculated gene frequency and genetic parameter, is shown in Table 2:
Table 2
Remarks: Na, number of alleles；Ne, effective number of allele；Ho observes heterozygosity；He, it is expected that heterozygosity；PIC,
Polymorphism information content.
Selected microsatellite locus allele is all at 3 or more as can be seen from Table 2, and polymorphism information content 0.2695~
0.8823, illustrate that selected microsatellite locus allele is more, polymorphism information content is abundant, adapt to carry out paternity test and
Germplasm genetic diversity analysis.
There are 7 sites that can effectively expand in hybridization porgy group, genetic parameter is all larger than black porgy group, shows filial generation
Has higher genetic diversity compared with black porgy.
After genotype data file establishes, Parentage determination, knot are carried out to 20 Eclectics porgys using the parent of known gender
Fruit is shown in Table 3:
Table 3
As can be seen that 20 individuals are successfully divided into different familys, wherein 6 individuals (HF1, HF3, HF4, HF7,
HF9, HF11) (being denoted as family 1) from a pair of of Parent (PR1, PB1), 4 individuals (HF2, HF5, HF8, HF10) (are denoted as house
It is 2) to share female parent PR3,2 individuals from another pair Parent (PR3, PB4), 2 individuals (HF14, HF16) and family 2
(HF15, HF18) and family 1 share male parent PB1, remaining individual is respectively from other Parents.
Sequence table:
The upstream primer sequence of SEQ ID NO.1 microsatellite marker primer 1
5'-AAGATGCTGCGCGATTCAAC-3'
The downstream primer sequence of SEQ ID NO.2 microsatellite marker primer 1
5'-ATCTGCTGTCGTACCCGATG-3'
The upstream primer sequence of SEQ ID NO.3 microsatellite marker primer 2
5'-CTGGCTGTTCAACTGCACAC-3'
The downstream primer sequence of SEQ ID NO.4 microsatellite marker primer 2
5'-CATTCTCCCACTCAACCCCC-3'
The upstream primer sequence of SEQ ID NO.5 microsatellite marker primer 3
5'-GCTGAACTGAGGTGGCTGAT-3'
The downstream primer sequence of SEQ ID NO.6 microsatellite marker primer 3
5'-AGCTTGAGCAGGACTGAACC-3'
The upstream primer sequence of SEQ ID NO.7 microsatellite marker primer 4
5'-TGGTCAGGTTTAGGCAGCTG-3'
The downstream primer sequence of SEQ ID NO.8 microsatellite marker primer 4
5'-GCCTCTCACACAGCTCTCTG-3'
The upstream primer sequence of SEQ ID NO.9 microsatellite marker primer 5
5'-AGTAGGTGATGGGCCGTACT-3'
The downstream primer sequence of SEQ ID NO.10 microsatellite marker primer 5
5'-CTCACCGTCAGCTCCACAAT-3'
The upstream primer sequence of SEQ ID NO.11 microsatellite marker primer 6
5'-ACACCAATGCTCTTCTCCGG-3'
The downstream primer sequence of SEQ ID NO.12 microsatellite marker primer 6
5'-AGATCGACCACAGACCTCCA-3'
The upstream primer sequence of SEQ ID NO.13 microsatellite marker primer 7
5'-ACAGTAGGGACAGGTCAGCT-3'
The downstream primer sequence of SEQ ID NO.14 microsatellite marker primer 7
5'-TCCTCTGGGGGACTCTGAAG-3'
The upstream primer sequence of SEQ ID NO.15 microsatellite marker primer 8
5'-TGTGCGTGTCCACAAGATGA-3'
The downstream primer sequence of SEQ ID NO.16 microsatellite marker primer 8
5'-TCAAGCTTGTGGACAGTGCA-3'
The upstream primer sequence of SEQ ID NO.17 microsatellite marker primer 9
5'-GCCTGCTCAAACATCTCAGC-3'
The downstream primer sequence of SEQ ID NO.18 microsatellite marker primer 9
5'-ACAATCACCTGACCTCCAGT-3'
The upstream primer sequence of SEQ ID NO.19 microsatellite marker primer 10
5'-GTGAATGTGCCACAGAGGGA-3'
The downstream primer sequence of SEQ ID NO.20 microsatellite marker primer 10
5'-GTGCAAACGCTTTGAGCAGA-3'
Sequence table
<110>Jiangsu Prov. Inst. of Marine Aquatic Products
<120>a kind of method for identifying hybridization porgy family using microsatellite marker
<160> 20
<170> SIPOSequenceListing 1.0
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Claims (5)
1. a kind of method for identifying hybridization porgy family using microsatellite marker, which comprises the steps of:
(1) clip red porgy raun and black porgy milter parent and its filial generation tail fin sample, carry out the extraction of genomic DNA；
(2) using parent in step (1) and the DNA of filial generation as template, multiplex PCR expansion is carried out using microsatellite marker primer
Increase；
(3) polyacrylamide gel electrophoresis, silver staining colour developing are carried out to the pcr amplification product of step (2), electrophorogram is used
The analysis of BandScan5.0 software, judges allele size in conjunction with PCR product length, determines idiotype；
(4) hybridization porgy and its genotype data file of parent are established according to the analysis result of step (3)；
(5) family differentiation is carried out using Cervus3.0 software.
2. utilizing the method for microsatellite marker identification hybridization porgy family as described in claim 1, which is characterized in that the microsatellite
Labeled primer has 10 groups, and the upstream primer sequence of microsatellite marker primer 1 is as shown in SEQ ID NO.1, and downstream primer sequence is such as
Shown in SEQ ID NO.2, marker site AS1；
The upstream primer sequence of microsatellite marker primer 2 is as shown in SEQ ID NO.3, downstream primer sequence such as SEQ ID NO.4
It is shown, marker site AS2；
The upstream primer sequence of microsatellite marker primer 3 is as shown in SEQ ID NO.5, downstream primer sequence such as SEQ ID NO.6
It is shown, marker site AS3；
The upstream primer sequence of microsatellite marker primer 4 is as shown in SEQ ID NO.7, downstream primer sequence such as SEQ ID NO.8
It is shown, marker site AS4；
The upstream primer sequence of microsatellite marker primer 5 is as shown in SEQ ID NO.9, downstream primer sequence such as SEQ ID NO.10
It is shown, marker site AS5；
The upstream primer sequence of microsatellite marker primer 6 is as shown in SEQ ID NO.11, downstream primer sequence such as SEQ ID
Shown in NO.12, marker site AS6；
The upstream primer sequence of microsatellite marker primer 7 is as shown in SEQ ID NO.13, downstream primer sequence such as SEQ ID
Shown in NO.14, marker site AS7；
The upstream primer sequence of microsatellite marker primer 8 is as shown in SEQ ID NO.15, downstream primer sequence such as SEQ ID
Shown in NO.16, marker site AS8；
The upstream primer sequence of microsatellite marker primer 9 is as shown in SEQ ID NO.17, downstream primer sequence such as SEQ ID
Shown in NO.18, marker site AS9；
The upstream primer sequence of microsatellite marker primer 1 is as shown in SEQ ID NO.19, downstream primer sequence such as SEQ ID
Shown in NO.20, marker site AS10.
3. utilizing the method for microsatellite marker identification hybridization porgy family as described in claim 1, which is characterized in that in step (2),
The reaction system (25 μ L) of PCR amplification are as follows: ddH2O 17.25 μ L, 10 × Buffer 2.5 μ L, dNTP 0.5 μ L, primer 2 μ L,
Genomic DNA 1 μ L, Mg2+1.5 μ L, 0.25 μ L of Taq enzyme.
4. utilizing the method for microsatellite marker identification hybridization porgy family as described in claim 1, which is characterized in that in step (2),
The reaction condition of PCR amplification are as follows: 94 DEG C of initial denaturation 10min；40s, 57.8-62.0 DEG C of annealing 40s of 94 DEG C of denaturation；72 DEG C of extensions
1min, 30 circulations；Last 72 DEG C of extensions 10min.
5. utilizing the method for microsatellite marker identification hybridization porgy family as claimed in claim 1 or 2 or 3 or 4, which is characterized in that
In step (3), the polyacrylamide gel electrophoresis using 8% non-denaturing polyacrylamide gel.
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