CN102534006A - Method for detecting homozygosis or heterozygosis of silkie fiber melanin gene - Google Patents
Method for detecting homozygosis or heterozygosis of silkie fiber melanin gene Download PDFInfo
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Abstract
The invention provides a method for detecting homozygosis or heterozygosis of a silkie fiber melanin gene, which comprises the following steps: carrying out mononucleotide polymorphic detection on a nucleotide of 10739109bp site of the No.20 chromosome (based on Chicken Genome Sequence Information, Edition WUGSC 2.1/galGal3, May 2006); and determining whether the silkie fiber melanin gene is homozygous or heterozygous according to the detection result. By selecting the homozygous/heterozygosis of the silkie Fm black property, the homozygote individual with Fm black property can be directly preserved, thereby greatly enhancing the breeding and preservation efficiency. The detection method provided by the invention is simple to operate, has the advantages of low cost and high accuracy, and can implement automatic detection.
Description
Technical field
The present invention relates to the Markers for Detection technology, specifically, relate to and a kind ofly detect that Gallus Domesticus fiber melanoma gene isozygotys or the method for heterozygosis.
Background technology
Along with Developing of Animal Industry, macroscopic features a breed of chicken with protect kind in occupy critical role, molecule marker also breeding with protect kind in played vital role.But the Study on Molecular Marker for the control macroscopic features is still less, only has a spot of macroscopic features to carry out assisted selection through molecule marker.
Gallus Domesticus has " perfect " characteristics such as black skin, black bone, black meat as the peculiar chicken kind of China.Wherein the black characteristic of Gallus Domesticus mainly receives fiber melanoma gene (Fm
+/ fm
-) influence.The reticular tissue black in color of Gallus Domesticus (Kuklenski, 1915), cockscomb, meat whisker are grape, and it is blue that ear, pin shin and beak are.Carry in the chicken kind of Fm gene, the position that melanochrome occurs comprises sarolemma, nerve, tendon, blood vessel, sexual gland, mesentery, periosteum and dermal layer of the skin etc.From to the attention of Gallus Domesticus " black look ", China utilizes the bone and the priming of muscle as traditional Chinese medicine of Gallus Domesticus, and some carry the improvement chicken kind of Fm gene, raises the food materials of arranging as tradition age in week to 12-14.
The black phenotype of Fm Gene Handling is a kind of dominant character of unit point control with respect to the non-black phenotype of fm Gene Handling; Thereby in the process of protecting kind and breeding; Can't accurately distinguish dominance homozygote and heterozygote individuality according to phenotype, cause the seed selection inefficiency.Along with the development of molecular amounts theory of heredity, need find the molecule marker that influences skin corium black proterties, through marker assisted selection, can identify the genotype that the candidate is individual quickly and accurately, fundamentally solve this guarantor and plant and the breeding problem.
Dna molecular marker is the genetic marker that is the basis with the nucleotide sequence variation in the genetic material between individuality, is the direct reflection of dna level genetic polymorphism.In the chicken genome, be distributed with a large amount of molecule markers; Carry huge genetic information amount; There have been a plurality of important characters to obtain the location at present, can have utilized relevant molecule marker to carry out the molecular marker assisted selection breeding, huge promoter action has been played in breeding.
(single nucleotide polymorphism SNP), mainly is meant on genomic level to be a kind of very good, effective molecule marker by the caused dna sequence polymorphism of the variation of single Nucleotide to SNP.
Summary of the invention
The purpose of this invention is to provide and a kind ofly detect that Gallus Domesticus fiber melanoma gene isozygotys or the method for heterozygosis.
In order to realize the object of the invention; The present invention provides a kind of and detects that Gallus Domesticus fiber melanoma gene isozygotys or the method for heterozygosis; It is that No. 20 karyomit(e) of Gallus Domesticus is positioned at the 10739109bp site (based on the chicken genome sequence Information page WUGSC 2.1/galGal3 of this shop; In May, 2006) Nucleotide carries out SNP and detects, and the fiber melanoma gene of judging Gallus Domesticus according to detected result is for isozygotying or heterozygosis.It is the tetra-sodium order-checking that SNP detects the preferred method that adopts.
The present invention detects also that Gallus Domesticus fiber melanoma gene isozygotys or the primer of heterozygosis, comprises F:5 '-GCCCCTTCCTGAAGCAATA-3 ' and R:5 '-CATGCAGGATCTCTATGAAGAAAA-3 '.
The present invention also provides and detects that Gallus Domesticus fiber melanoma gene isozygotys or the probe of heterozygosis, and its nucleotides sequence classifies 5 as '-GTATTCTAGAAGCCATTCC-3 '.
The present invention also provides the detection Gallus Domesticus fiber melanoma gene that contains above-mentioned primer and probe to isozygoty or the test kit of heterozygosis.
The present invention further provides a kind of and detects that Gallus Domesticus fiber melanoma gene isozygotys or the method for heterozygosis; It is that the genomic dna with Gallus Domesticus is a template; Adopt above-mentioned primer to carry out pcr amplification; With above-mentioned probe amplified production is carried out SNP then and detect, the fiber melanoma gene of judging Gallus Domesticus according to detected result is for isozygotying or heterozygosis; Wherein, to detect be the Nucleotide that is positioned at the 10739109bp site to No. 20 karyomit(e) to SNP.
The PCR reaction system is: genomic dna 30ng, 1x amplification buffer, Mg
2+50mM, dNTPs 5mM, primers F 10 μ M, primer R 10 μ M, Taq archaeal dna polymerase 1U adds water and mends to 25 μ l.
The PCR reaction conditions is: 95 ℃ of sex change 5min; 95 ℃ of sex change 15sec, 57 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 45 circulations; 72 ℃ are extended 7min; 20 ℃ of preservations.
Detecting the genotype that No. 20 karyomit(e) of chicken to be measured is positioned at the 10739109bp site is AG or GG (A is an adenine nucleotide, and G is the bird pyrimidine nucleotide); When genotype is AG; A: G=1: 1, represent that then all there is CNV (gene copy number variation) in this site on two karyomit(e)s, on every karyomit(e) the genotype of a copy being arranged all is AA; The genotype of a copy is GG, and chicken to be measured is that Fm black dominance is isozygotied; A: G=1: 2 o'clock, represent that then only there is CNV in this site on a karyomit(e), the genotype of a copy is AA; The genotype of a copy is GG, does not have CNV on another karyomit(e), has only a copy; Genotype is GG, and chicken to be measured is a Fm black dominance heterozygosis; If the genotype of chicken to be measured is GG, represent that then there is not CNV in this site on two karyomit(e)s, all have only a copy on every karyomit(e), genotype is GG, chicken to be measured is that fm non-black recessiveness is isozygotied.
Wherein, the detection method of SNP detection employing is the Pyrosequencing tetra-sodium sequencing technologies of PyroMark ID quantitative inheritance analytical system (Biotage company).
Aforesaid method, primer are to can be applicable to a breed of chicken with probe, test kit.
Method provided by the present invention and product (primer, probe, test kit), to the Fm black proterties of Gallus Domesticus isozygoty/heterozygosis selects, can directly Fm black proterties homozygote individuality be remained, thereby improve breeding widely and protect kind of an efficient.The present invention provides an efficiently and accurately, simple and rapid molecular genetic marker for the molecular marker assisted selection of chicken breeding, for the seed selection of the Fm black proterties of chicken with protect a kind of effective molecular marker breeding means that kind provide.With this genetic marker the Fm black proterties of chicken is carried out marker assisted selection; Can alleviate actual breeding and the problem that can't distinguish Fm black homozygote and heterozygote of protecting in planting effectively; Provide seed selection to protect kind of an efficient; And lay the foundation for utilizing this proterties to carry out the molecule aggregation breeding, will accelerate breeding process.Detection method of the present invention is simple to operate, cost, and accuracy is high, can realize that robotization detects.Develop the relevant detection test kit according to the method for the invention, can be used for selecting to carry the beneficial gene type (AG, A: G=1: individuality 1), protect kind of work for a breed of chicken and facilitate.
Description of drawings
Fig. 1 detects SNP (genotype AG, the A: G=1: 1) that individual No. 20 karyomit(e) of Gallus Domesticus of homozygote is positioned at the 10739109bp site for Pyrosequencing tetra-sodium sequence measurement of the present invention.
Fig. 2 detects SNP (genotype AG, the A: G=1: 2) that individual No. 20 karyomit(e) of Gallus Domesticus of heterozygote is positioned at the 10739109bp site for Pyrosequencing tetra-sodium sequence measurement of the present invention.
Fig. 3 detects the SNP (genotype GG) that No. 20 karyomit(e) of non-black proterties chicken is positioned at the 10739109bp site for Pyrosequencing tetra-sodium sequence measurement of the present invention.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize, the conventional means that used technique means is well known to those skilled in the art among the embodiment, used test materials and reagent are the commercial goods." % " that relates among the embodiment like no specified otherwise, is mass percent.
Confirming of the foundation of embodiment 1 Pyrosequencing tetra-sodium order-checking detection method and polymorphic site
1 gene type assay
1.1 family makes up: make up and be specifically designed to the experimental population that the research location influences the fiber melanoma gene (Fm) of chicken reticular tissue melanin deposition; Construction process is: F0 is 1 of silk plumage Gallus Domesticus cock and 3 of Youxi fiber crops chicken hens for the parent; Adopt the method for artificial insemination, go into to incubate simultaneously after waiting to collect 30 pieces of eggs; F1 for hatching after, all chick are raised by ordinary method under same raising condition, treat sexual maturity after; It is dark therefrom to select 3 skin black level, and the close cock of blackness, and every cock and 10 Youxi fiber crops chicken hens are formed family; Form 3 familys altogether; Adopt the method for artificial insemination, go into to incubate after whenever collecting 120 pieces of eggs, go into to incubate two batches altogether; F2 for hatching after, all chick are raised to 12 ages in week by ordinary method under same raising condition.
1.2 experiment material: this experiment has been chosen F0, F1 and the F2 of 3 familys of Fm family for individuality, amounts to 245.In the Fm family, write down F0, F1 and F2 cockscomb, pin shin, various reticular tissue and skin corium skin color for individuality.
1.3 the extraction of genomic dna
Chicken is the wing venous blood collection during 12 ages in week, and the back cracking is handled in anti-freezing, and behind protease K digesting, with the imitative extracting of phenol, TE dissolves-20 ℃ of preservations.
1.4 pcr amplification
Genomic dna to extract is a template, with primer the fragment (Seq ID No.4) of striding No. 20 karyomit(e) 10739109bp site (based on the chicken genome sequence Information page WUGSC 2.1/galGal3 of this shop, in May, 2006) is carried out pcr amplification.
F：5′-GCCCCTTCCTGAAGCAATA-3′
R：5′-biotin-CATGCAGGATCTCTATGAAGAAAA-3′
PCR reaction system (25 μ l) final concentration is: genomic dna 30ng, 1x amplification buffer, Mg
2+50mM, dNTPs 5mM, F 10 μ M, R 10 μ M, Taq Gold archaeal dna polymerase 1U adds aqua sterilisa and mends to 25 μ l.
PCR reaction conditions: 95 ℃ of sex change 5min; 95 ℃ of sex change 15sec, 57 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 45 circulations; 72 ℃ are extended 7min; 20 ℃ of preservations.
1.5 the PCR product detects
Get 5 μ l PCR products and carry out the detection of 2% sepharose.
1.6 tetra-sodium order-checking
Using P yroMark ID quantitative inheritance analytical system is a template with the PCR product, with primer the tetra-sodium order-checking is carried out in the 10739109bp site.The probe that uses is:
S：5′-GTATTCTAGAAGCCATTCC-3′
1.6.1 sample pretreatment, strand separation and purification operation
1) before use, guarantees that all solution all reach room temperature;
2) in PSQ 96 plates, add the annealing primer that 45 μ l contain 0.3 μ M sequencing primer in advance, add 10pM primer 2 μ l generally speaking;
3) use Vertex mixing Sepharose beads;
4) the sepharoe beads total amount that will use (every sample 3 μ l) is transferred in the Eppendorf pipe;
5) in sepharose beads, add binding buffer liquid, make average each sample that the volume of 50 μ l arranged approximately, the mixture mixing;
6) above mixture is added in the PCR product (50 μ l reaction volume) every sample 50 μ l;
7) with PCR product mixing 10 minutes at normal temperatures, make beads combine with vitamin H, for than long segment, but the proper extension mixing time;
8) in Vacuum prep workstation, add 180ml high purity water, 70% ethanol, lavation buffer solution and 120ml sex change damping fluid in four sample panel successively;
9) open the pump of vacuum prep workstation, vacuum prep tool was cleaned in high purity water 30 seconds; Then vacuum prep tool is moved on in the PCR plate, grasp sepharose beads (please combine with the PCR product to accomplish this operation in back three minutes, do not let Beads sink to the pipe end again) at beads; Pick up the PCR plate, whether most of beads has been attracted on the vacuum prep tool in inspection;
10) vacuum prep tool was put into 70% ethanol 5 seconds;
11) move on in the sex change damping fluid 5 seconds then;
12) move on to again cleaning 5-10 second in the lavation buffer solution;
13) be placed on the corresponding top of containing the plate hole of sequencing primer to suction nozzle, do not contact liquid level, turn off pump;
14) vacuum prep tool is put into the plate that contains sequencing primer, shake, discharge sepharose beads (sequencing primer also can add at last)
15) use high purity water to clean vacuum prep tool;
PSQ 96 plates that 16) will be placed with sample be placed on be heated on the ThermoPlate 80 ℃ 2 minutes, cool to room temperature can carry out the Pyrosequencing reaction again.
1.6.2 machine on the sample
1) opens PyroMark ID main frame;
2) open computer and the corresponding software of controlling;
3) select SNP, SNP Runs and New SNP Run (or selecting SQA);
4) insert Run name, select Instrument parameters;
5) selection needs the sample well of use, and clicks Activate;
6) in Entry, insert SNP entry (or SQA);
7) be ready to sample, and add in the sample panel;
8) prepare enzyme and substrate, and all reagent were at room temperature placed 10 minutes before use;
9) enzyme, substrate and dNTPs are added in the reagent cabin;
The PSQ 96 Plate Low that 10) will be placed with the process sample pretreatment of sample are placed on the instrument;
11) examining instrument parameter selects;
12) click the Run button, operation;
13) analytical results
Produce three kinds of genotype:
First kind of genotype: AG, A: G=1: 1, chicken to be measured is isozygotied for Fm black;
Second kind of genotype: AG, A: G=1: 2, chicken to be measured is a Fm black heterozygosis;
The third genotype: GG, chicken to be measured is isozygotied for the fm non-black.
2 correlation analysiss
The result is as shown in table 1; This result shows that in 245 individuals that detected, (A: G=1: 1) genotype has 2 to AG; (A: G=1: 2) genotype has 106 to AG; The GG genotype has 137, and (A: G=1: 1) (A: G=1: 2) the genotype individuality is Fm black proterties phenotype to AG, and GG genotype individuality is fm non-black proterties phenotype for genotype individuality and AG.Wherein (A: G=1: 1) the genotype individuality is the dominance homozygote to AG, and (A: G=1: 2) the genotype individuality is goneoclin to AG, and GG genotype individuality is allozygote.
The correlation analysis statistics of No. 20 karyomit(e) 10739109bp site different genotype of table 1 and Fm black proterties
| Phenotype | Quantity | AG (A: G=1: 1) genotype | AG (A: G=1: 2) genotype | The GG genotype |
| Fm black proterties | 108 | 2 | 106 | 0 |
| Fm non-black proterties | 137 | 0 | 0 | 137 |
| Amount to | 245 | 2 | 106 | 137 |
The application of embodiment 2 molecule markers
Respectively 2 kinds of 3 kinds (available from Poultry Institute, Chinese Academy of Agricultural Science) with Fm black phenotype and fm non-black phenotype are measured, concrete kind is seen table 2 and table 3.
1.1 the extraction of genomic dna
Chicken is the wing venous blood collection during 12 ages in week, and the back cracking is handled in anti-freezing, and behind protease K digesting, with the imitative extracting of phenol, TE dissolves-20 ℃ of preservations.
1.2 pcr amplification
Genomic dna to extract is a template, with primer the fragment of striding No. 20 karyomit(e) 10739109bp site is carried out pcr amplification.
F：5′-GCCCCTTCCTGAAGCAATA-3′
R：5′-biotin-CATGCAGGATCTCTATGAAGAAAA-3′
PCR reaction system (25 μ l) final concentration is: genomic dna 30ng, 1x amplification buffer, Mg
2+50mM, dNTPs 5mM, primers F 10 μ M, primer R 10 μ M, Taq Gold archaeal dna polymerase 1U adds aqua sterilisa and mends to 25 μ l.
PCR reaction conditions: 95 ℃ of sex change 5min; 95 ℃ of sex change 15sec, 57 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 45 circulations; 72 ℃ are extended 7min; 20 ℃ of preservations.
1.3 the PCR product detects
Get 5 μ l PCR products and carry out the detection of 2% sepharose.
1.4 tetra-sodium order-checking
Using P yroMark ID quantitative inheritance analytical system, with the PCR product be template, with primer tetra-sodium order-checking is carried out in the 10739109bp site.The probe that uses is:
S：5′-GTATTCTAGAAGCCATTCC-3′
1.4.1 sample pretreatment, strand separation and purification operation
1) before use, guarantees that all solution all reach room temperature;
2) in PSQ 96 plates, add the annealing primer that 45 μ l contain 0.3 μ M sequencing primer in advance, add 10pM primer 2 μ l generally speaking;
3) use Vertex mixing Sepharose beads;
4) the sepharoe beads total amount that will use (every sample 3 μ l) is transferred in the Eppendorf pipe;
5) in sepharose beads, add binding buffer liquid, make average each sample that the volume of 50 μ l arranged approximately, the mixture mixing;
6) above mixture is added in the PCR product (50 μ l reaction volume) every sample 50 μ l;
7) with PCR product mixing 10 minutes at normal temperatures, make beads combine with vitamin H, for than long segment, but the proper extension mixing time;
8) in Vacuum prep workstation, add 180ml high purity water, 70% ethanol, lavation buffer solution and 120ml sex change damping fluid in four sample panel successively;
9) open the pump of vacuum prep workstation, vacuum prep tool was cleaned in high purity water 30 seconds; Then vacuum prep tool is moved on in the PCR plate, grasp sepharose beads (please combine with the PCR product to accomplish this operation in back three minutes, do not let Beads sink to the pipe end again) at beads; Pick up the PCR plate, whether most of beads has been attracted on the vacuum prep tool in inspection;
10) vacuum prep tool was put into 70% ethanol 5 seconds;
11) move on in the sex change damping fluid 5 seconds then;
12) move on to again cleaning 5-10 second in the lavation buffer solution;
13) be placed on the corresponding top of containing the plate hole of sequencing primer to suction nozzle, do not contact liquid level, turn off pump;
14) vacuum prep tool is put into the plate that contains sequencing primer, shake, discharge sepharose beads (sequencing primer also can add at last)
15) use high purity water to clean vacuum prep tool;
PSQ 96 plates that 16) will be placed with sample be placed on be heated on the ThermoPlate 80 ℃ 2 minutes, cool to room temperature can carry out the Pyrosequencing reaction again.
1.4.2 machine on the sample
1) opens PyroMark ID main frame;
2) open computer and the corresponding software of controlling;
3) select SNP, SNP Runs and New SNP Run (or selecting SQA);
4) insert Run name, select Instrument parameters;
5) selection needs the sample well of use, and clicks Activate;
6) in Entry, insert SNP entry (or SQA);
7) be ready to sample, and add in the sample panel;
8) prepare enzyme and substrate, and all reagent were at room temperature placed 10 minutes before use;
9) enzyme, substrate and dNTPs are added in the reagent cabin;
The PSQ 96 Plate Low that 10) will be placed with the process sample pretreatment of sample are placed on the instrument;
11) examining instrument parameter selects;
12) click the Run button, operation;
13) analytical results
Produce two kinds of genotype:
First kind of genotype: AG, A: G=1: 1, chicken to be measured is isozygotied for Fm black;
Second kind of genotype: GG, chicken to be measured is isozygotied for the fm non-black.
1.5 correlation analysis result
From table 2, can find out, in the kind of 3 Fm black phenotypes, can detect Fm black genotype in 90 individuals, wherein (A: G=1: 1) genotype is a homozygote to AG, amounts to 82 individuals; (A: G=1: 2) genotype is a heterozygote to AG, amounts to 8 individuals.Can find out that from table 3 in the kind of 2 fm non-black phenotypes, 60 individuals are the GG genotype.
Show that this site can be used as a genetic marker, be applied to the seed selection of chicken Fm black proterties homozygous individual and protect in kind of the work raising efficiency of selection.
The correlation analysis statistics of No. 20 karyomit(e) 10739109bp site different genotype of table 2 and Fm black phenotype kind
| Fm black kind | Quantity | AG (A: G=1: 1) genotype | AG (A: G=1: 2) genotype | The GG genotype |
| Silk |
30 | 30 | 0 | 0 |
| Jinhu County black- |
30 | 30 | 0 | 0 |
| Fast big black- |
30 | 25 | 5 | 0 |
| Amount to | 90 | 85 | 5 | 0 |
The correlation analysis statistics of No. 20 karyomit(e) 10739109bp site different genotype of table 3 and Fm non-black phenotype kind
| Fm non-black kind | Quantity | AG (A: G=1: 1) genotype | AG (A: G=1: 2) genotype | The GG genotype |
| Stealthy Cold boiled |
30 | 0 | 0 | 30 |
| |
30 | 0 | 0 | 30 |
| Amount to | 60 | 0 | 0 | 60 |
The application of embodiment 3 molecular marker breedings
Identify 31 of the chickens of the dark skin that ground peasant households such as Wuding County, Yunnan, Tengchong raise scattered, choose through present method be accredited as Fm black proterties heterozygous genes type AG (A: G=1: each 1 of cock 2) and hen, as F0 for breeding.Produce 16 F1 for individuality; (A: G=1: each 1 of cock 1) and hen carry out mating will wherein to be accredited as Fm black proterties homozygous genotype AG; Produce 14 F2 and be dark skin, be Fm black proterties homozygous genotype AG (A: G=1: 1) through identifying for individuality.Concrete outcome is seen table 4-table 6.
1.1 the extraction of genomic dna
Chicken is the wing venous blood collection during 12 ages in week, and the back cracking is handled in anti-freezing, and behind protease K digesting, with the imitative extracting of phenol, TE dissolves-20 ℃ of preservations.
1.2 pcr amplification
Genomic dna to extract is a template, with primer the fragment of striding No. 20 karyomit(e) 10739109bp site is carried out pcr amplification.
F：5′-GCCCCTTCCTGAAGCAATA-3′
R：5′-biotin-CATGCAGGATCTCTATGAAGAAAA-3′
PCR reaction system (25 μ l) final concentration is: genomic dna 30ng, 1x amplification buffer, Mg
2+50mM, dNTPs 5mM, primers F 10 μ M, primer R 10 μ M, Taq Gold archaeal dna polymerase 1U adds aqua sterilisa and mends to 25 μ l.
PCR reaction conditions: 95 ℃ of sex change 5min; 95 ℃ of sex change 15sec, 57 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 45 circulations; 72 ℃ are extended 7min; 20 ℃ of preservations.
1.3 the PCR product detects
Get 5 μ l PCR products and carry out the detection of 2% sepharose.
1.4 tetra-sodium order-checking
Using P yroMark ID quantitative inheritance analytical system, with the PCR product be template, with primer tetra-sodium order-checking is carried out in the 10739109bp site.The probe that uses is:
S：5′-GTATTCTAGAAGCCATTCC-3′
1.4.1 sample pretreatment, strand separation and purification operation
1) before use, guarantees that all solution all reach room temperature;
2) in PSQ 96 plates, add the annealing primer that 45 μ l contain 0.3 μ M sequencing primer in advance, add 10pM primer 2 μ l generally speaking;
3) use Vertex mixing Sepharose beads;
4) the sepharoe beads total amount that will use (every sample 3 μ l) is transferred in the Eppendorf pipe;
5) in sepharose beads, add binding buffer liquid, make average each sample that the volume of 50 μ l arranged approximately, the mixture mixing;
6) above mixture is added in the PCR product (50 μ l reaction volume) every sample 50 μ l;
7) with PCR product mixing 10 minutes at normal temperatures, make beads combine with vitamin H, for than long segment, but the proper extension mixing time;
8) in Vacuum prep workstation, add 180ml high purity water, 70% ethanol, lavation buffer solution and 120ml sex change damping fluid in four sample panel successively;
9) open the pump of vacuum prep workstation, vacuum prep tool was cleaned in high purity water 30 seconds; Then vacuum prep tool is moved on in the PCR plate, grasp sepharose beads (please combine with the PCR product to accomplish this operation in back three minutes, do not let Beads sink to the pipe end again) at beads; Pick up the PCR plate, whether most of beads has been attracted on the vacuum prep tool in inspection;
10) vacuum prep tool was put into 70% ethanol 5 seconds;
11) move on in the sex change damping fluid 5 seconds then;
12) move on to again cleaning 5-10 second in the lavation buffer solution;
13) be placed on the corresponding top of containing the plate hole of sequencing primer to suction nozzle, do not contact liquid level, turn off pump;
14) vacuum prep tool is put into the plate that contains sequencing primer, shake, discharge sepharose beads (sequencing primer also can add at last)
15) use high purity water to clean vacuum prep tool;
PSQ 96 plates that 16) will be placed with sample be placed on be heated on the ThermoPlate 80 ℃ 2 minutes, cool to room temperature can carry out the Pyrosequencing reaction again.
1.4.2 machine on the sample
1) opens PyroMark ID main frame;
2) open computer and the corresponding software of controlling;
3) select SNP, SNP Runs and New SNP Run (or selecting SQA);
4) insert Run name, select Instrument parameters;
5) selection needs the sample well of use, and clicks Activate;
6) in Entry, insert SNP entry (or SQA);
7) be ready to sample, and add in the sample panel;
8) prepare enzyme and substrate, and all reagent were at room temperature placed 10 minutes before use;
9) enzyme, substrate and dNTPs are added in the reagent cabin;
The PSQ 96 Plate Low that 10) will be placed with the process sample pretreatment of sample are placed on the instrument;
11) examining instrument parameter selects;
12) click the Run button, operation;
13) analytical results
Produce three kinds of genotype:
First kind of genotype: AG, A: G=1: 1, chicken to be measured is isozygotied for Fm black;
Second kind of genotype: AG, A: G=1: 2, chicken to be measured is a Fm black heterozygosis;
The third genotype: GG, chicken to be measured is isozygotied for the fm non-black.
1.5 correlation analysis result
Can find out that from table 4 in the individuality of 31 Fm black phenotypes, (A: G=1: 1) genotypic Fm black homozygote is 9 for AG; (A: G=1: 2) genotypic Fm black heterozygote is 22 for AG.Can find out that from table 5 in the individuality in 16 F1 generations, (A: G=1: 1) genotypic Fm black homozygote is 4 for AG; (A: G=1: 2) genotypic Fm black heterozygote is 9 for AG.3 of the genotypic fm non-black of GG homozygotes.Can find out that from table 6 in the individuality in 14 F2 generations, (A: G=1: 1) genotypic Fm black homozygote is 14 for AG; (A: G=1: 2) genotypic Fm black heterozygote is 0 for AG.0 of the genotypic fm non-black of GG homozygote.
Experiment showed, through present method, in actual breeding, have Fm black proterties homozygous genotype AG (A: G=1: chicken 1) has accurately, characteristics easily for choosing.
The F0 of No. 20 karyomit(e) 10739109bp site different genotype of table 4 and Yunnan Fm black phenotype adds up for the chicken correlation analysis
| The Fm black individual | Quantity | AG (A: G=1: 1) genotype | AG (A: G=1: 2) genotype | The GG genotype |
| Cock | 14 | 4 | 10 | 0 |
| Hen | 17 | 5 | 12 | 0 |
| Amount to | 31 | 9 | 22 | 0 |
No. 20 karyomit(e) 10739109bp site different genotype of table 5 and Yunnan F1 are for the correlation analysis statistics of hybridization chicken colour of skin phenotype
| Individual | Quantity | AG (A: G=1: 1) genotype | AG (A: G=1: 2) genotype | The GG genotype |
| Fm black proterties cock | 6 | 2 | 4 | 0 |
| Fm non-black proterties cock | 1 | 0 | 0 | 1 |
| Fm black proterties hen | 7 | 2 | 5 | 0 |
| Fm non-black proterties hen | 2 | 0 | 0 | 2 |
| Amount to | 16 | 4 | 9 | 3 |
No. 20 karyomit(e) 10739109bp site different genotype of table 6 and Yunnan F2 are for hybridization black phenotype chicken correlation analysis statistics
| The Fm black individual | Quantity | AG (A: G=1: 1) genotype | AG (A: G=1: 2) genotype | The GG genotype |
| Cock | 6 | 6 | 0 | 0 |
| Hen | 8 | 8 | 0 | 0 |
| Amount to | 14 | 14 | 0 | 0 |
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (9)
1. one kind is detected that Gallus Domesticus fiber melanoma gene isozygotys or the method for heterozygosis; It is characterized in that; The Nucleotide that No. 20 karyomit(e) of Gallus Domesticus is positioned at the 10739109bp site carries out SNP and detects, and the fiber melanoma gene of judging Gallus Domesticus according to detected result is for isozygotying or heterozygosis.
2. method according to claim 1 is characterized in that, it is the tetra-sodium order-checking that SNP detects the method that is adopted.
3. detect that Gallus Domesticus fiber melanoma gene isozygotys or the primer of heterozygosis, comprising:
F:5 '-GCCCCTTCCTGAAGCAATA-3 '; With
R：5′-CATGCAGGATCTCTATGAAGAAAA-3′。
4. detecting that Gallus Domesticus fiber melanoma gene isozygotys or the probe of heterozygosis, its nucleotides sequence classifies 5 as '-GTATTCTAGAAGCCATTCC-3 '.
5. detect that Gallus Domesticus fiber melanoma gene isozygotys or the test kit of heterozygosis, it contains described primer of claim 3 and the described probe of claim 4.
6. one kind is detected that Gallus Domesticus fiber melanoma gene isozygotys or the method for heterozygosis; It is characterized in that; Genomic dna with Gallus Domesticus is a template; Adopt the described primer of claim 3 to carry out pcr amplification, with the described probe of claim 4 amplified production carried out SNP then and detect, according to the fiber melanoma gene of detected result judgement Gallus Domesticus for isozygotying or heterozygosis;
Wherein, to detect be the Nucleotide that is positioned at the 10739109bp site to No. 20 karyomit(e) to SNP.
7. method according to claim 6 is characterized in that, it is the tetra-sodium order-checking that SNP detects the method that is adopted.
8. according to claim 6 or 7 described methods, it is characterized in that the PCR reaction system is: genomic dna 30ng, 1x amplification buffer, Mg
2+50mM, dNTPs 5mM, primers F 10 μ M, primer R 10 μ M, Taq archaeal dna polymerase 1U adds water and mends to 25 μ l.
9. according to claim 6 or 7 described methods, it is characterized in that the PCR reaction conditions is: 95 ℃ of sex change 5min; 95 ℃ of sex change 15sec, 57 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 45 circulations; 72 ℃ are extended 7min; 20 ℃ of preservations.
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| CN106086172A (en) * | 2016-06-13 | 2016-11-09 | 江苏省家禽科学研究所 | A kind of chicken protects the method that population at individual apolegamy optimizes |
| CN110029175A (en) * | 2019-04-22 | 2019-07-19 | 广西大学 | A kind of breeding method using molecular labeling quickly breeding black silk plumage Gallus domesticlus brisson |
| CN111876492A (en) * | 2020-07-15 | 2020-11-03 | 华南农业大学 | Molecular marker influencing skin color of shank of chicken and application thereof |
| CN113322333A (en) * | 2021-07-06 | 2021-08-31 | 广西大学 | CNV molecular marker combination related to Guangxi hemp chicken body size and slaughter traits based on whole genome sequencing screening and application |
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| CN105603094B (en) * | 2016-02-22 | 2019-01-04 | 杭州市农业科学研究院 | A kind of relevant SNP marker of black-bone chicken colour of skin character and application |
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| CN113322333B (en) * | 2021-07-06 | 2022-07-05 | 广西大学 | CNV molecular marker combination related to Guangxi hemp chicken body size and slaughter traits based on whole genome sequencing screening and application |
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