CN103122384A - Breeding method of laying hen with low egg yolk cholesterol content - Google Patents
Breeding method of laying hen with low egg yolk cholesterol content Download PDFInfo
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Abstract
The invention discloses a breeding method of a laying hen with low egg yolk cholesterol content. The breeding method comprises the following steps of (1) taking a DNA (deoxyribonucleic acid) sequence including chicken HMGCR (Hydroxymethyl glutaric acyl coenzyme) gene as a template, designing a pair of primers, and carrying out sample chicken individual gene parting by a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method; (2) utilizing a genetic effect analysis model to determine selected and remained genetype; (3) building a family from individual bodies of cocks and hens according with the selected and remained genetype; (4) extracting the DNA in later generation of the selected and remained family hen, carrying out PCR-RFLP analysis on the pair of primers in step (1), selecting and remaining the hens with selected and remained genetype in step (2); and (5) repeating steps (3) and (4) to continuously breed for at least three generations, so as to obtain the laying hen with low egg yolk cholesterol content. The HMGCR gene molecular marker is adopted to assist and select the breeding method; the generation interval is shortened; the breeding accuracy is improved; the breeding flock feeding capacity is reduced; and the feeding cost is also reduced.
Description
Technical field
The present invention relates to the selection of a kind of low cholesterol level of vitellus laying hen, belong to bird Biotechnology in Genetic Breeding field.
Background technology
Cholesterol is the important component part of the various meinbranous structures of zooblast and neural myelin, is the precursor of bile acide, steroid hormone and Vitamin D3 500,000 I.U/GM, has important physiological function.If but body take in a large amount of cholesterol and can cause that in blood, its content obviously raises, and this causes one of important factor that the cardiovascular disordeies such as coronary sclerosis, cerebral thrombosis occur just.American heart research association recommends the every day cholesterol intake should be lower than 300 milligrams as far back as the beginning of the seventies in last century.Dietary cholesterol derives from animal food, and egg is higher a kind of of cholesterol level in animal food.Studies show that, the cholesterol in egg is the important factor that affects total cholesterol in human plasma (TC) and low density lipoprotein cholesterol (LDL-CHOL) level.Consider from diet and the angle healthy, that improve the cultivation level of improving people, reduce cholesterol content in eggs and have very important significance.The method that reduces at present cholesterol in egg mainly contains two kinds: the one, and the laying hen new lines of selecting to hang down cholesterol level of vitellus by the conventional breeding approach; The 2nd, regulate and control by feed nutrition, namely adopt in diet the additive that adds some trophicity, non-nutritive or fat-reducing medicament etc.But the employing traditional breeding method, because the period of going through is long, Advances in Breeding is slow, requires great effort, takes fund; Adopt the mode of feed nutrition regulation and control, though successful wherein additive or drug residue problem and production performance and sanitary impact also be can not be ignored.Therefore, solve the high problem of cholesterol level of vitellus, the most basic approach is still by the low cholesterol level of vitellus laying hen new lines of modern molecular breeding technology seed selection.
Summary of the invention
The selection that the purpose of this invention is to provide a kind of low cholesterol level of vitellus laying hen, the egg yolk cholesterol level lasts for a long time solve to reduce, there are residual problem in additive or medicine.
In order to realize above purpose, the technical solution adopted in the present invention is to provide the selection of a kind of low cholesterol level of vitellus laying hen, comprises the following steps:
1) take the DNA sequence dna that comprises chicken HMGCR gene as template, the design pair of primers utilizes the PCR-RFLP method to carry out sample chicken individuals gene type; The PCR-RFLP endonuclease bamhi is a fragment, and clip size is 162bp, called after TT type; The PCR-RFLP endonuclease bamhi is two fragments, and clip size is respectively 141bp and 21bp, called after GG type; The PCR-RFLP endonuclease bamhi is three fragments, and clip size is respectively 162bp, 141bp and 21bp, called after GT type;
2) utilize the genetic effect analytical model, different genotype and sample chicken individuals cholesterol level of vitellus and shell egg cholesterol level are carried out association analysis, relatively the cholesterol level of vitellus of different genotype, determine the genotype of selecting and remain;
3) will meet the genotypic male and female chicken individuals of selecting and remain and set up family, breeding is of future generation;
4) extract family chicken offspring's the DNA that selects and remain, carry out PCR-RFLP with the pair of primers in step 1) and analyze, selecting and remain wherein is step 2) in the genotypic chicken of selecting and remain;
5) repeating step 3) and 4) subculture seed selection three generations at least, low cholesterol level of vitellus laying hen obtained.
Described primer is:
Forward primer: 5'-CATTGCCTGTGGTCAGGT-3';
Reverse primer: 5'-GCAGTTAGAGCTGCCTAGATT-3'.
Described PCR reaction system is: ultrapure water 9.5 μ L, each 1 μ L of the upstream and downstream primer of 10pmol/ μ L, the DNA profiling 1 μ L of 50ng/ μ L, 2 * Taq Master Mix12.5 μ L, reaction cumulative volume 25 μ L.
Described PCR response procedures is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 30s, 57.7 ℃ of annealing 30s, 72 ℃ are extended 30s, and last 72 ℃ are fully extended 10min, and 4 ℃ of preservations, wherein sex change, annealing, extension are totally 30 circulations.
The described PCR-RFLP enzyme system of cutting is: Hinf I restriction endonuclease 0.3 μ L, 10 * PCR Buffer, 2 μ L, PCR product 10 μ L, ultrapure water 7.7 μ L, reaction cumulative volume 20 μ L.
Described genetic effect analytical model: Y=μ+G+E, wherein: Y is the observed value that Egg Quality is measured; μ is colony's mean value; G is the genotype fixed effect; E is random error.
The described genotype of selecting and remain is the GG type.
The concrete grammar of described step 3) is: adopt the closed population selection-breeding method to carry out pure line selection, set up 50 of familys, male and female ratio 1:10, individual cage was raised to 40 ages in week, record the egg number of every hen, 40 weeks average egg weight and the cholesterol level of vitellus during age, calculates the average of egg number, egg size and 3 proterties of cholesterol level of vitellus of 50 whole hens of family and each family hen; Take cholesterol level of vitellus as major traits, take into account egg size and egg number, the cholesterol level of vitellus of selecting and remain is lower than 50 whole hen averages of family, and egg size and egg productivity are higher than front 13 familys of 50 whole hen averages of family, and breeding is of future generation.
The laying hen that the inventive method selects has good production performance and egg quality, and the comparable previous level of its cholesterol level of vitellus reduces more than 20%, meets the human consumer for the health requirements of egg consumption.The present invention adopts HMGCR gene molecule marker assisted selection method, shortens the generation interval, improves the seed selection accuracy, reduces the breeding group number of animals raised, reduces feeding cost.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure of chicken genomic dna;
Fig. 2 is the agarose gel electrophoresis figure of chicken HMGCR gene PCR amplified production;
Fig. 3 is that chicken HMGCR gene PCR product enzyme is cut polymorphic electrophorogram;
Fig. 4 is chicken different genotype sequencer map.
Embodiment
1, the extraction of sample chicken DNA
In the underlying group of breeding, choose 500 single cages of sample chicken and raise, extract the DNA of sample chicken, step is as follows:
1) DNA extraction
A) add respectively 500 μ L TE damping fluids in 80 μ L whole bloods, SDS lysate (25%) 12 μ L and Proteinase K (10mg/L) 10 μ L, concussion mixing 30min, 57 ℃ of water-bath digestion are spent the night;
B) add the saturated phenol 602 μ L of Tris, vibration shakes up 10min, the centrifugal 10min of 10,000r/min;
C) get supernatant liquor, add the saturated phenol 500 μ L of Tris, vibration shakes up 10min, the centrifugal 10min of 10,000r/min;
D) get supernatant liquor, add equal-volume phenol/chloroform/primary isoamyl alcohol (25:24:1), slowly shake up 10min, the centrifugal 10min of 1,2000r/min;
E) get supernatant liquor, add equal-volume chloroform/primary isoamyl alcohol (24:1), slowly shake up 10min, the centrifugal 10min of 1,0000r/min;
F) get supernatant liquor, add 2 times of volumes to be chilled in advance-20 ℃ of dehydrated alcohols, concussion 10min, the centrifugal 10min of 10,000r/min;
G) abandon supernatant liquor, add 70% ethanol 500 μ L concussion 10min, the centrifugal 15min of 10,000r/min repeats twice; Be inverted seasoning, add appropriate TE solution dissolving DNA.
2) DNA detection
A) gel maker is cleaned up, insert comb;
B) weigh the 0.5g agarose, put into Erlenmeyer flask, add 1 * TBE60mL, the microwave oven moderate heat is heated to without bubble;
C) add EB solution in Erlenmeyer flask, making its final concentration is 0.5 μ g/mL, the jog mixing; Solution is cooled to 60 ℃, pours in gel maker;
D) after the complete cooled and solidified of solution, take out comb, gel is changed in electrophoresis chamber; Add 1 * tbe buffer liquid in electrophoresis chamber, liquid level exceeds gel 5mm;
E) with unified loading after DNA sample 2 μ L and sample-loading buffer 2 μ L mixings; After 120V voltage electrophoresis 30min, utilize uv analyzer to observe.
Measure the DNA sample at the OD value at 260nm, 280nm place, calculating DNA content and OD with ultraviolet spectrophotometer
260/ OD
280Ratio; Greater than 1.8, explanation has the existence of RNA, need carry out purifying as ratio; As degrade and obviously again extract respective sample DNA.
The agarose detected result of genomic dna of the present invention is seen Fig. 1.As can be seen from Figure 1, the quality of the chicken genomic dna of the present invention's extraction is very high.
2, pcr amplification
1) design of primer is with synthetic
Chicken 3-hydroxy-3-methylglutaric acid list acyl coenzyme A reductase enzyme (HMGCR) gene order (accession number: NC_006127.2) as template take the Genebank announcement, utilize Oligo6.71 and Primer Premier5.0 primer-design software at the interior design primer of HMGCR gene extron 17, as follows:
Forward primer: 5'-CATTGCCTGTGGTCAGGT-3'(SEQ ID NO:1)
Reverse primer: 5'-GCAGTTAGAGCTGCCTAGATT-3'(SEQ ID NO:2)
2) pcr amplification
PCR reaction system such as table 1.
Table 1PCR reaction system
| | Volume | |
| 2×Taq?Master?Mix | 12.5μL | |
| Upstream primer (10pmol/ μ L) | 1μL | |
| Downstream primer (10pmol/ μ L) | 1μL | |
| DNA profiling (50ng/ μ L) | 1μL | |
| Ultrapure water | 9.5μL | |
| Cumulative volume | 25μL |
PCR response procedures such as table 2.
Table 2PCR amplification program
4 ℃ of preservations of pcr amplification product.
Pcr amplification product is carried out the agarose gel electrophoresis analysis, the results are shown in Figure 2.As can be seen from Figure 2, the pcr amplification product clip size is 162bp, with the in the same size of Design Theory, therefore can prove successfully to amplify chicken HMGCR gene fragment.
3, PCR-RFLP analyzes
1) enzyme is cut digestion reaction program such as table 3.
Table 3 restriction enzyme digestion system
| Component | Volume |
| 10×PCR?Buffer | 2μL |
| The PCR product | 10μL |
| Restriction enzyme Hinf I | 0.3μL |
| Ultrapure water | 7.7μL |
| Cumulative volume | 20.0μL |
2) agarose gel electrophoresis analysis
PCR product enzyme is cut polymorphic electrophorogram, as shown in Figure 3.Cut the clip size of product according to enzyme in image, artificial judgment statistics genotype.When the PCR-RFLP endonuclease bamhi is a fragment, clip size is 162bp, called after TT type; When the PCR-RFLP endonuclease bamhi is two fragments, clip size is respectively 141bp and 21bp, called after GG type; When the PCR-RFLP endonuclease bamhi is three fragments, clip size is respectively 162bp, 141bp and 21bp, called after GT type.Respectively different genotype is checked order, result as shown in Figure 4.As can be seen from Figure 4, the 12217bp place of HMGCR gene is polymorphic site.
4, Determination of Cholesterol Content
Under the chromatographic condition of selecting, the retention time of cholesterol is 9min.Be respectively 0.05 with cholesterol concentration, 0.10,0.20,0.30,0.40,0.50mg/mL standard cholesterol working fluid loading, applied sample amount is 5 μ L, and take peak area as ordinate zou Y, cholesterol concentration is X-coordinate X drawing standard curve, the line linearity of going forward side by side returns, regression equation is Y=2258X+12.33, coefficient R
2=0.999.In this colony in egg yolk in cholesterol average content and shell egg the cholesterol average content be respectively 13.00mg/g yolk and 437.28mg/100g shell egg.
5, the correlation analysis of different genotype and cholesterol level of vitellus and shell egg cholesterol level
Utilize the genetic effect analytical model, the correlation analysis result with different genotype and cholesterol level of vitellus and shell egg cholesterol level the results are shown in Table 4.The genetic effect analytical model:
Y=μ+G+E
Wherein, Y is the observed value that Egg Quality is measured; μ is colony's mean value; G is the genotype fixed effect; E is random error.
Table 4HMGCR gene is g.12217G〉T site and cholesterol level association analysis
| Proterties | GG(LSM±S.E.) | GT(LSM±S.E.) | TT(LSM±S.E.) | P-value |
| Cholesterol Content in Egg Yolk (mg/g yolk) | 11.643±0.935 | 13.639±0.381 | 13.704±1.167 | 0.139 |
| Shell egg cholesterol level (mg/100g shell egg) | 391.291±32.643 b | 467.401±13.287 a | 453.162±40.712 a | 0.099 |
Annotate: numerical value is mean number ± standard error; Letter identical table differential is different not remarkable, and the lowercase alphabet differential is different significantly, and is not remarkable without mark expression difference.
As can be seen from Table 4, g.12217G〉on the T site, different genotype is on cholesterol level impact remarkable (P＜0.1) in egg, wherein the individual cholesterol level of vitellus of GG type (mg/g yolk) respectively and GT type individual low 15.03% more individual than TT type and the individual shell egg cholesterol level of 14.66%, GG type (mg/100g shell egg) respectively and GT type individuality more individual than TT type hang down 13.65% and 16.28%.Concerning the laying hen strain of the low cholesterol level of vitellus of seed selection, g.12217G〉T can be used as molecule marker, and the GG type is the genotype of selecting and remain.
Adopt the closed population selection-breeding method to carry out pure line selection the genotypic sample chicken of selecting and remain in embodiment 1, set up 50 of familys, male and female ratio 1:10, individual cage was raised to 40 ages in week, record the egg number of every hen, 40 weeks average egg weight and the cholesterol level of vitellus during age, calculates the average of egg number, egg size and 3 proterties of cholesterol level of vitellus of 50 whole hens of family and each family hen, see Table 5.Take cholesterol level of vitellus as major traits, take into account egg size and egg number, the cholesterol level of vitellus of selecting and remain is lower than colony's average, and egg size and egg productivity are higher than front 13 familys of colony's average, and breeding is of future generation, sees Table 6.
The average of egg number, egg size and 3 proterties of cholesterol level of vitellus of the whole hens of table 550 family and each family hen
| Proterties | The minimum average of family | The highest average of family | Colony's average |
| Egg number (individual) | 90.82 | 94.23 | 92.45 |
| Egg size (g) | 50.71 | 53.64 | 52.32 |
| Cholesterol level of vitellus (mg/g yolk) | 10.74 | 12.37 | 11.82 |
Wherein, colony's average is the average of 50 whole hens of family.
The select and remain average of egg number, egg size and 3 proterties of cholesterol level of vitellus of family of table 6
| Proterties | The minimum average of family | The highest average of family | Colony's average |
| Egg number (individual) | 92.51 | 93.83 | 93.08 |
| Egg size (g) | 52.33 | 53.64 | 53.11 |
| Cholesterol level of vitellus (mg/g yolk) | 10.74 | 11.69 | 11.38 |
Wherein, colony's average is the average of 50 whole hens of family.
Extract family chicken offspring's in 2 age in week the DNA that selects and remain in embodiment 2, carry out PCR-RFLP with the pair of primers in embodiment 1 and analyze, according to the HMGCR gene molecule marker of candidate's individuality g.12217G T, the GG type of selecting and remain carries out the early molecule marker assisted selection.
The GG type individuality of selecting and remain in embodiment 3 is further selected with embodiment 2,3 mode, in 3 generations of subculture seed selection, obtains low cholesterol level of vitellus laying hen.
The yolk cholesterol average content of the low cholesterol level of vitellus laying hen of embodiment 4 has dropped to the 9.94mg/g yolk of 3 generations from the 13.00mg/g yolk of 0 generation after measured, and fall is 23.54%.
Claims (8)
1. the selection of a low cholesterol level of vitellus laying hen, is characterized in that, comprises the following steps:
1) take the DNA sequence dna that comprises chicken HMGCR gene as template, the design pair of primers utilizes the PCR-RFLP method to carry out sample chicken individuals gene type; The PCR-RFLP endonuclease bamhi is a fragment, and clip size is 162bp, called after TT type; The PCR-RFLP endonuclease bamhi is two fragments, and clip size is respectively 141bp and 21bp, called after GG type; The PCR-RFLP endonuclease bamhi is three fragments, and clip size is respectively 162bp, 141bp and 21bp, called after GT type;
2) utilize the genetic effect analytical model, different genotype and sample chicken individuals cholesterol level of vitellus and shell egg cholesterol level are carried out association analysis, relatively the cholesterol level of vitellus of different genotype, determine the genotype of selecting and remain;
3) will meet the genotypic male and female chicken individuals of selecting and remain and set up family, breeding is of future generation;
4) extract family chicken offspring's the DNA that selects and remain, carry out PCR-RFLP with the pair of primers in step 1) and analyze, selecting and remain wherein is step 2) in the genotypic chicken of selecting and remain;
5) repeating step 3) and 4) subculture seed selection three generations at least, low cholesterol level of vitellus laying hen obtained.
2. the selection of a kind of low cholesterol level of vitellus laying hen according to claim 1, is characterized in that, described primer is:
Forward primer: 5'-CATTGCCTGTGGTCAGGT-3';
Reverse primer: 5'-GCAGTTAGAGCTGCCTAGATT-3'.
3. the selection of a kind of low cholesterol level of vitellus laying hen according to claim 1, it is characterized in that, described PCR reaction system is: ultrapure water 9.5 μ L, each 1 μ L of the upstream and downstream primer of 10pmol/ μ L, the DNA profiling 1 μ L of 50ng/ μ L, 2 * Taq Master Mix12.5 μ L, reaction cumulative volume 25 μ L.
4. the selection of a kind of low cholesterol level of vitellus laying hen according to claim 1, it is characterized in that, described PCR response procedures is: 95 ℃ of denaturation 5min, 95 ℃ of sex change 30s, 57.7 ℃ annealing 30s, 72 ℃ are extended 30s, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations, wherein sex change, annealing, extension are totally 30 circulations.
5. the selection of a kind of low cholesterol level of vitellus laying hen according to claim 1, is characterized in that, the described PCR-RFLP enzyme system of cutting is: Hinf I restriction endonuclease 0.3 μ L, 10 * PCR Buffer, 2 μ L, PCR product 10 μ L, ultrapure water 7.7 μ L, reaction cumulative volume 20 μ L.
6. the selection of a kind of low cholesterol level of vitellus laying hen according to claim 1, is characterized in that, described genetic effect analytical model: Y=μ+G+E, and wherein: Y is the observed value that Egg Quality is measured; μ is colony's mean value; G is the genotype fixed effect; E is random error.
7. the selection of a kind of low cholesterol level of vitellus laying hen according to claim 1, is characterized in that, the described genotype of selecting and remain is the GG type.
8. the selection of a kind of low cholesterol level of vitellus laying hen according to claim 1, it is characterized in that, the concrete grammar of described step 3) is: adopt the closed population selection-breeding method to carry out pure line selection, set up 50 of familys, male and female ratio 1:10, individual cage was raised to 40 ages in week, record the egg number of every hen, 40 weeks average egg weight and the cholesterol level of vitellus during age, calculates the average of egg number, egg size and 3 proterties of cholesterol level of vitellus of 50 whole hens of family and each family hen; Take cholesterol level of vitellus as major traits, take into account egg size and egg number, the cholesterol level of vitellus of selecting and remain is lower than 50 whole hen averages of family, and egg size and egg productivity are higher than front 13 familys of 50 whole hen averages of family, and breeding is of future generation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103416351A (en) * | 2013-09-11 | 2013-12-04 | 山西省农业科学院畜牧兽医研究所 | Method for establishing Bian chicken quick-grow type AA strain and slow-grow type GG strain colony |
| CN103563851A (en) * | 2013-11-20 | 2014-02-12 | 河南农业大学 | Method for cultivating dominant-green-shin white feather broiler strain based on molecular assistant selection |
-
2013
- 2013-01-23 CN CN201310024771XA patent/CN103122384A/en active Pending
Non-Patent Citations (2)
| Title |
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| 汪仕奎等: "蛋鸡的胆固醇代谢调控研究进展", 《动物营养学报》 * |
| 邓呈逊: "鸡OVR基因PCR-RFLP检测及其与产蛋性能相关性的研究", 《安徽农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103416351A (en) * | 2013-09-11 | 2013-12-04 | 山西省农业科学院畜牧兽医研究所 | Method for establishing Bian chicken quick-grow type AA strain and slow-grow type GG strain colony |
| CN103563851A (en) * | 2013-11-20 | 2014-02-12 | 河南农业大学 | Method for cultivating dominant-green-shin white feather broiler strain based on molecular assistant selection |
| CN103563851B (en) * | 2013-11-20 | 2015-04-22 | 河南农业大学 | Method for cultivating dominant-green-shin white feather broiler strain based on molecular assistant selection |
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Application publication date: 20130529 |