CN103436630A - High-quality broiler chicken inosinic acid and intramuscular fat content related gene polymerization breeding method - Google Patents
High-quality broiler chicken inosinic acid and intramuscular fat content related gene polymerization breeding method Download PDFInfo
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Abstract
The invention discloses a high-quality broiler chicken inosinic acid and intramuscular fat content related gene polymerization breeding method comprising the following steps: respectively amplifying a gene by using three pairs of primers under the condition of PCR (Polymerase Chain Reaction) by taking two-continuous-generation high-quality broiler chicken genome DNAs (Deoxyribose Nucleic Acids) as templates; judging the sizes of amplification products of the first primer, the second primer and the third primer according to agarose gel electrophoresis, and then, screening the mutation of three alkali bases by using a DNA sequencing technology, wherein the three alkali bases include an exon 9 of an adenylosuccinate lyase gene, a non-coding region of a phosphoribosylglycineamide synthetase-5-aminoimidazole ribonucleotide synthetase-phosphoribosylglycineamide transmethylase gene 5' and an intron 2 of an adipocyte fatty acid binding protein gene; and then, carrying out gene typing and gene frequency analysis on SNPs (Single Nucleotide Polymorphisms) of the three loci by using polyacrylamide gel electrophoresis. By using the high-quality broiler chicken inosinic acid and intramuscular fat content related gene polymerization breeding method, the favorable polymerization of a protogene type can be realized, and meanwhile, the genetic mechanisms of the chicken meat quality, flavor and character can be searched more comprehensively.
Description
Technical field
The present invention relates to a kind of selection, particularly the selection of a kind of high quality meat chicken t-inosinic acid and the polymerization of intramuscular fat content genes involved.
Technical background
Improve the important directions that has become the high quality meat chicken breeding research with the raising meat flavor, wherein, t-inosinic acid and intramuscular fat content are the important factors that affects the meat flavor proterties.But t-inosinic acid and intramuscular fat all are subject to various factors, the conventional herd breeding progress is slower; Therefore utilize the correlation candidate gene of t-inosinic acid and intramuscular fat proterties, it is current comparatively effectively means that the application molecular marker assisted selection is accelerated genetic progress, thereby carries out chicken matter local flavor breeding improvement from hereditary level.The research of the screening of meat quality trait molecular marker and marker assisted selection possessed to certain basis both at home and abroad, especially some monogenic character marker assisted selection have been made some progress.But Meat Quality is a Comprehensive Traits that is subject to controlled by multiple genes, only from single-gene, starts with and carries out marker assisted selection, often can not reach comprehensive improvement meat, the purpose of finally cultivating high-quality chicken.
Summary of the invention
Technical problem to be solved by this invention is to provide the selection of a kind of high quality meat chicken t-inosinic acid and the polymerization of intramuscular fat content genes involved, and it realizes the favourable polymerization of preponderant genotype, can more comprehensively seek the genetic mechanism of chicken matter local flavor proterties simultaneously.
The present invention solves above-mentioned technical problem by following technical proposals: the selection of a kind of high quality meat chicken t-inosinic acid and the polymerization of intramuscular fat content genes involved, it is characterized in that, and it comprises the following steps:
Step 1, continuous two the generation high quality meat chicken genomic dnas of take are masterplate, under the PCR condition, utilize the first primer, the second primer, three-primer, increase respectively ADSL, GARS-AIRS-GART, A-FABP gene;
Step 2, according to agarose gel electrophoresis, the first primer, the second primer, three-primer amplified production being carried out to size judges, then adopt the DNA sequencing technology screening to adenosine succsinic acid lyase gene exon 9, phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene 5 ' non-coding region, and adipocyte fatty acid-binding protein gene intron 2 has the sudden change of three bases;
Step 3, the recycling polyacrylamide gel electrophoresis carries out gene type and gene frequency analysis to the SNPs in these three sites, analyze the individual polymorphism of high quality meat chicken and the relation of t-inosinic acid and intramuscular fat content and the relation between different genotype combination and t-inosinic acid and intramuscular fat content, and trace back parental generation, follow the tracks of filial generation, detect the genotype combination of high quality meat chicken individuality and the relation of t-inosinic acid and intramuscular fat content, analyze the contribution of different genotype in t-inosinic acid and intramuscular fat content proterties form, select the genotype combination of high t-inosinic acid and intramuscular fat content individuality, analyze the apolegamy mode that high t-inosinic acid and intramuscular fat content idiotype are combined to form, apply the multiple gene polymerization core group that these apolegamy modes form the high t-inosinic acid of high quality meat chicken and intramuscular fat content.
Preferably, described the first primer is as follows: upstream primer 1F:5 '-TCTCCTTCCACAGGCTCAA-3 '; Downstream primer 1R:5 '-AACTGCTGCACTGCACGAT-3 '; Described the second primer P2 is as follows: upstream primer 2F:5 '-ACAGTTGCCAGTCTGATTA-3 '; Downstream primer 2R:5 '-CATCGCCAGAGTTAGAAGT-3 '; Described three-primer P3 is as follows: upstream primer 3F:5 '-GGAATGTGACAACGCTAA-3 '; Downstream primer 3R:5 '-CGGATAAGGAGGGCAGAG-3 '.
Preferably, described the first primer amplification adenosine succsinic acid lyase gene exon 9, sudden change for screening high-quality broiler chicken adenosine succsinic acid lyase gene site, the second primer amplification phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene 5 ' non-coding region, sudden change for screening high-quality broiler chicken phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene locus, three-primer amplification adipocyte fatty acid-binding protein gene intron 2, sudden change for screening high-quality broiler chicken adipocyte fatty acid-binding protein gene site.
The present invention has following positively effect: the present invention realizes the favourable polymerization of preponderant genotype, can more comprehensively seek the genetic mechanism of chicken matter local flavor proterties simultaneously.
Embodiment
For content of the present invention is more likely to be clearly understood, below according to specific embodiment, the present invention is further detailed explanation.
Gene pyramiding is that the beneficial gene be dispersed in different varieties is aggregated in same genome, in segregating generation, by molecule marker, selects the individuality that contains a plurality of target genes, realizes the polymerization of beneficial gene.Adopt the gene pyramiding means can realize molecular breeding truly.Therefore study the development and utilization that the assembly effect of a plurality of meat candidate genes will be accelerated indigenous chicken kind elite germplasm characteristic, for the industry development of high-quality chicken provides technical support.
It is research object that ADSL, GARS-AIRS-GART, A-FABP gene are take in the present invention, result is in eight kinds of polymeric type, and the CCAAMM genotype chest muscle imp content (3.077mg/g) after favourable single-gene polymerization is significantly higher than other non-complete beneficial gene type polymerization individuality.Except the CCBBMM type, the intramuscular fat content of CCAAMM type is significantly higher than other polymerization genotype (P<0.05).By the preliminary identification of gene pyramiding effect, there are assembly effect in ADSL, GART, A-FABP gene, and the polymerization genotype can be used as the effective candidate's mark of Daheng's high quality meat chicken Meat Quality.Gene pyramiding colony can suitably adjust expansion, is conducive to the individual screening of more preponderant genotypes.Molecular marker assisted selection is to carry out a kind of effective way of gene pyramiding breeding, and simultaneously, the gene pyramiding breeding is feasible in the high quality meat chicken molecular breeding.
The selection of high quality meat chicken t-inosinic acid of the present invention and the polymerization of intramuscular fat content genes involved comprises the following steps:
Step 1, continuous two the generation high quality meat chicken genomic dnas of take are masterplate, in the PCR(polymerase chain reaction, PolymeraseChainReaction) under condition, utilize three couples of primer P1, P2 and P3(the first primer P1, the second primer P2, three-primer P3), increase respectively ADSL, GARS-AIRS-GART, A-FABP gene, wherein:
Described the first primer P1 is as follows: upstream primer 1F:5 '-TCTCCTTCCACAGGCTCAA-3 '; Downstream primer 1R:5 '-AACTGCTGCACTGCACGAT-3 ';
Described the second primer P2 is as follows: upstream primer 2F:5 '-ACAGTTGCCAGTCTGATTA-3 '; Downstream primer 2R:5 '-CATCGCCAGAGTTAGAAGT-3 ';
Described three-primer P3 is as follows: upstream primer 3F:5 '-GGAATGTGACAACGCTAA-3 '; Downstream primer 3R:5 '-CGGATAAGGAGGGCAGAG-3 ';
The first primer P1 amplification adenosine succsinic acid lyase (ADSL) gene extron 9, for the sudden change in screening high-quality broiler chicken adenosine succsinic acid lyase gene site;
Second primer P2 amplification phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase (GARS-AIRS-GART) gene 5 ' non-coding region, for the sudden change of screening high-quality broiler chicken phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene locus;
Three-primer P3 amplification adipocyte fatty acid binding protein (A-FABP) gene intron 2, for the sudden change in screening high-quality broiler chicken adipocyte fatty acid-binding protein gene site;
Step 2, according to agarose gel electrophoresis, three pairs of primer extension products being carried out to size judges, then adopt the DNA sequencing technology screening to adenosine succsinic acid lyase gene exon 9, phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene 5 ' non-coding region, and adipocyte fatty acid-binding protein gene intron 2 has the sudden change of three bases;
Step 3, the recycling polyacrylamide gel electrophoresis carries out gene type and gene frequency analysis to the SNPs in these three sites, analyze the individual polymorphism of high quality meat chicken and the relation of t-inosinic acid and intramuscular fat content and the relation between different genotype combination and t-inosinic acid and intramuscular fat content, and trace back parental generation, follow the tracks of filial generation, detect the genotype combination of high quality meat chicken individuality and the relation of t-inosinic acid and intramuscular fat content, analyze the contribution of different genotype in t-inosinic acid and intramuscular fat content proterties form, select the genotype combination of high t-inosinic acid and intramuscular fat content individuality, analyze the apolegamy mode that high t-inosinic acid and intramuscular fat content idiotype are combined to form, apply the multiple gene polymerization core group that these apolegamy modes form the high t-inosinic acid of high quality meat chicken and intramuscular fat content.
The condition of described pcr amplification is: 10 μ L amplification systems: 2 * Long Taq PCR MasterMix, 5 μ L; DdH2O, 3.5 μ L; Upstream and downstream primer (F, R) (10pmol/ul), each 0.5 μ L; DNA profiling (50ng/ μ L), 0.5 μ L;
The PCR response procedures is as follows: 1) utilize the PCR response procedures of primer P1 to be: 95 ℃ of denaturation 3min; 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 50s, carry out altogether 35 circulations; 72 ℃ are extended 10min, 4 ℃ of preservations.2) utilize the PCR response procedures of primer P2 to be: 95 ℃ of denaturation 3min; 95 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 50s, carry out altogether 35 circulations; 72 ℃ are extended 10min, 4 ℃ of preservations.3) utilize the PCR response procedures of primer P1 to be: 95 ℃ of denaturation 3min; 95 ℃ of sex change 30s, 59 ℃ of annealing 30s, 72 ℃ are extended 50s, carry out altogether 35 circulations; 72 ℃ are extended 10min, 4 ℃ of preservations.
There is the sudden change of C/T in described adenosine succsinic acid lyase gene exon 9 at 10191bp, phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-there is the sudden change of C/T in phosphoribosyl glycinamide methylferase gene 5 ' non-coding region at 153bp, and there is the sudden change of C/T in adipocyte fatty acid-binding protein gene intron 2 at 1684bp;
Described gene type is that result is as follows with the PCR product of 12% polyacrylamide gel electrophoresis determination and analysis primer P1, P2 and P3:
The first primer P1 site: the base of homozygous CC in the 10191bp position is C, and the base of heterozygous CT in the 10191bp position is C/T, and the base of homozygous TT in the 10191bp position is T;
The second primer P2 site: the base of homozygous AA in the 153bp position is C, and the base of heterozygous AB in the 153bp position is C/T, and the base of homozygous BB in the 153bp position is T;
Three-primer P3 site: the base of homozygous MM in the 1684bp position is C, and the base of heterozygous MN in the 1684bp position is C/T, and the base of homozygous NN in the 1684bp position is T;
Described favourable single-gene aggregate combinations is: CCAAMM(F0 generation and F1 generation);
The contribution rate of described different genotype in imp content and intramuscular fat content proterties form is, the individual imp content of CCAAMM genotype after favourable single-gene polymerization (3.077mg/g) is significantly higher than other non-complete beneficial gene type polymerization individuality, high by 7.96% than genotype CC, high by 9.91% than frequency of genotypes AA,, high by 12.21% than genotype MM; Except the CCBBMM type, the intramuscular fat content of CCAAMM type is significantly higher than other polymerization genotype, high by 5.49% than genotype CC, high by 5.68% than frequency of genotypes AA,, high by 6.11% than genotype MM.
Utilize primer P1, P2 and P3 respectively to adenosine succsinic acid lyase gene exon 9, phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene 5 ' non-coding region and the pcr amplification of adipocyte fatty acid-binding protein gene intron 2 and the detection of polymorphism thereof.
1, the sampling and processing of high quality meat chicken blood sample
From vein blood sampling 2mL under Daheng's high quality meat chicken wing, inject vacuum test tube (EDTAK2 anti-freezing) with syringe.Ice chest is preserved, and takes back laboratory.The centrifugal 10min of 3000r/min, 4 ℃ save backup.
The present embodiment adopts 300 parts of high quality meat chicken blood samples, picks up from poultry breeding company limited of Sichuan Daheng.
2, the extraction of blood sample genomic dna, purifying
(1) blood sample is got 10 μ L through centrifugal after removing serum, adds PBS solution dilution to 250 μ L, after add in 500 μ L bacterial lysate Buffer AP1(1.5mL centrifuge tubes), vortex vibration 10s;
(2) add 100 μ L albumen precipitation liquid Buffer AP2, vortex vibration 10s, the centrifugal 10min of 12000g;
(3) get supernatant liquor 600 μ L, add (preparative column is placed in the 2mL centrifuge tube in advance) in the DNA preparative column, the centrifugal 1min of 12000g, abandon filtrate;
(4) add 700 μ L washings Buffer W1A in preparative column, room temperature is placed 2min, and the centrifugal 1min of 12000g, abandon filtrate;
(5) to adding the 800 μ L liquid Buffer W2 that desalts in preparative column, the centrifugal 1min of 12000g, abandon filtrate; Add 500 μ L Buffer W2, the centrifugal 1min of 12000g, abandon filtrate again; The centrifugal 1min of 12000g;
(6) preparative column is placed in to the 1.5mL centrifuge tube, in silica film central authorities, adds 200 μ L Buffer TE(to be preheating to 65 ℃), the standing 1min of room temperature; The centrifugal 1min eluted dna of 12000g.
Genomic dna detects: adopt 1.5% agarose gel electrophoresis and ultraviolet spectrophotometry to detect DNA purity and concentration.
1.5% sepharose configuration: take agarose 0.225g, add 15mL0.5 * TBE solution, Microwave Dissolution, then add 0.75 μ L Gold View nucleic acid dye, glue (horizontal glue); Electrophoresis: mix with 1 μ L 6 * DNA Loading Buffer 5 μ L DNA are molten, loading, under the 120V condition in Horizontal electrophoresis tank in 0.5 * TBE electrophoretic buffer electrophoresis 30min, detect the DNA electrophoretic band, gel imaging system is taken a picture and is preserved.
Ultraviolet spectrophotometer is measured nucleic acid: measure nucleic acid ultraviolet absorptivity value at 260nm and 280nm place, OD260, OD280 and OD260/OD280 value, detect DNA concentration and purity, guarantees to pollute without protein, RNA and small-molecule substance.
Mutational site is detected, and adopts native polyacrylamide gel electrophoresis (PAGE), and concrete steps are as follows:
(1) 12% polyacrylamide gel configuration: measure 30% acrylamide/methene acrylamide premixed liquid (29:1) 8mL, 1 * TBE solution 12mL, add 10% ammonium persulphate 120 μ L, TEMED 24 μ L, and in mixing on ice, rapid encapsulating (vertical glue).
(2) PCR product denaturation: 20 μ L LIS-SSCP sample-loading buffers and 1 μ L PCR product are mixed, 97 ℃ of sex change 2min, room temperature is placed.
(3) electrophoresis: get 10 μ L PCR denatured products, loading, under the 120V condition in Vertial electrophorestic tank in 1 * TBE electrophoretic buffer ice bath electrophoresis 2～4h, detect the single strand conformation polymorphism of sex change PCR product.
(4) silver dyes: the glue shovel is beaten easily lower gel, distilled water washing 30s; Add the about 30r/min of 100mL 0.1%AgNO3 staining fluid dyeing 10min(shaking table), then wash 10s with distilled water; Adopt the colour developing of 100mL sodium tetraborate nitrite ion, add formaldehyde 500 μ L jog colour developing 10min to obtaining promising result, twice of distilled water washing; Gel imaging system is taken a picture and is preserved.
According to the native polyacrylamide gel electrophoresis result, the PCR product of selecting the different genotype individuality is served extra large Invitrogen company and is carried out sequencing after the AxyPrep DNA gel reclaims test kit (Axygen company) purifying.
Experimentation is first to utilize polyacrylamide gel electrophoresis to screen adenosine succsinic acid lyase gene exon 9, phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene 5 ' non-coding region, and adipocyte fatty acid-binding protein gene intron 2 has the sudden change of three bases; Adopt again the DNA sequencing technology to be verified these three SNPs site different genotype, then carry out gene type and genotype frequency analysis.
The above specific embodiment; technical problem, technical scheme and beneficial effect to solution of the present invention further describe; institute is understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any modification of making, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (3)
1. the selection of a high quality meat chicken t-inosinic acid and the polymerization of intramuscular fat content genes involved, is characterized in that, it comprises the following steps:
Step 1, continuous two the generation high quality meat chicken genomic dnas of take are masterplate, under the PCR condition, utilize the first primer, the second primer, three-primer, increase respectively ADSL, GARS-AIRS-GART, A-FABP gene;
Step 2, according to agarose gel electrophoresis, the first primer, the second primer, three-primer amplified production being carried out to size judges, then adopt the DNA sequencing technology screening to adenosine succsinic acid lyase gene exon 9, phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene 5 ' non-coding region, and adipocyte fatty acid-binding protein gene intron 2 has the sudden change of three bases;
Step 3, the recycling polyacrylamide gel electrophoresis carries out gene type and gene frequency analysis to the SNPs in these three sites, analyze the individual polymorphism of high quality meat chicken and the relation of t-inosinic acid and intramuscular fat content and the relation between different genotype combination and t-inosinic acid and intramuscular fat content, and trace back parental generation, follow the tracks of filial generation, detect the genotype combination of high quality meat chicken individuality and the relation of t-inosinic acid and intramuscular fat content, analyze the contribution of different genotype in t-inosinic acid and intramuscular fat content proterties form, select the genotype combination of high t-inosinic acid and intramuscular fat content individuality, analyze the apolegamy mode that high t-inosinic acid and intramuscular fat content idiotype are combined to form, apply the multiple gene polymerization core group that these apolegamy modes form the high t-inosinic acid of high quality meat chicken and intramuscular fat content.
2. the selection of high quality meat chicken t-inosinic acid and the polymerization of intramuscular fat content genes involved according to claim 1, is characterized in that, described the first primer is as follows: upstream primer 1F:5 '-TCTCCTTCCACAGGCTCAA-3 '; Downstream primer 1R:5 '-AACTGCTGCACTGCACGAT-3 '; Described the second primer P2 is as follows: upstream primer 2F:5 '-ACAGTTGCCAGTCTGATTA-3 '; Downstream primer 2R:5 '-CATCGCCAGAGTTAGAAGT-3 '; Described three-primer P3 is as follows: upstream primer 3F:5 '-GGAATGTGACAACGCTAA-3 '; Downstream primer 3R:5 '-CGGATAAGGAGGGCAGAG-3 '.
3. the selection of high quality meat chicken t-inosinic acid and the polymerization of intramuscular fat content genes involved according to claim 1, it is characterized in that, described the first primer amplification adenosine succsinic acid lyase gene exon 9, sudden change for screening high-quality broiler chicken adenosine succsinic acid lyase gene site, the second primer amplification phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene 5 ' non-coding region, sudden change for screening high-quality broiler chicken phosphoribosyl-glycinamide synthetase-5-amino-imidazole ribotide synthetic enzyme-phosphoribosyl glycinamide methylferase gene locus, three-primer amplification adipocyte fatty acid-binding protein gene intron 2, sudden change for screening high-quality broiler chicken adipocyte fatty acid-binding protein gene site.
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| CN104673902A (en) * | 2015-02-10 | 2015-06-03 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker related to breast muscle weight and breast muscle percentage of chicken and application of SNP molecular marker |
| CN106701935A (en) * | 2016-12-20 | 2017-05-24 | 江苏省家禽科学研究所 | Fast pyramiding breeding method of high-quality broiler multi-target traits |
| CN110241235A (en) * | 2019-07-26 | 2019-09-17 | 湖南农业大学 | ADSL gene and genetic marking method as XuPu breeder geese inosine acid content |
| CN110468213A (en) * | 2019-07-31 | 2019-11-19 | 扬州大学 | A kind of black chicken inosinicacid of gold thatch and the relevant molecular labeling of intramuscular fat content and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104673902A (en) * | 2015-02-10 | 2015-06-03 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker related to breast muscle weight and breast muscle percentage of chicken and application of SNP molecular marker |
| CN106701935A (en) * | 2016-12-20 | 2017-05-24 | 江苏省家禽科学研究所 | Fast pyramiding breeding method of high-quality broiler multi-target traits |
| CN106701935B (en) * | 2016-12-20 | 2020-01-07 | 江苏省家禽科学研究所 | Rapid multi-target character polymerization breeding method for high-quality broiler chickens |
| CN110241235A (en) * | 2019-07-26 | 2019-09-17 | 湖南农业大学 | ADSL gene and genetic marking method as XuPu breeder geese inosine acid content |
| CN110241235B (en) * | 2019-07-26 | 2022-06-21 | 湖南农业大学 | ADSL gene and genetic marking method for its content of \28294u-goose inosinic acid |
| CN110468213A (en) * | 2019-07-31 | 2019-11-19 | 扬州大学 | A kind of black chicken inosinicacid of gold thatch and the relevant molecular labeling of intramuscular fat content and application |
| CN110468213B (en) * | 2019-07-31 | 2022-06-17 | 扬州大学 | Molecular marker related to contents of inosinic acid and intramuscular fat in golden black chicken and application of molecular marker |
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