CN102084847A - Method for culturing high-quality efficient stress-resistant complete set line of large white pigs - Google Patents
Method for culturing high-quality efficient stress-resistant complete set line of large white pigs Download PDFInfo
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Abstract
The invention relates to a method for culturing a high-quality efficient stress-resistant complete set line of large white pigs, which comprises the following steps of: building a basic group based on gene identification and disease resistance verification of F18 colibacillosis resistant major genes FUT1M307 of a core group, and culturing the high-quality efficient stress-resistant complete set line of the large white pigs by combining immunology principle, molecular breeding technology and conventional breeding means and using a candidate gene strategy. The complete set line pigs have the advantages of stress resistance, strong environmental adaptability, weaned piglet diarrhea and edema disease resistance, high lean meat rate, excellent meat quality, high feed reward and high birth number.
Description
Technical field
The invention belongs to Animal Genetics, Breeding and Reproduction, technical field of molecular biology, be specifically related to the breeding method of the supporting system of high-quality and efficient Large White resistance.
Background technology
Since the establishment of the nation, particularly since the reform and opening-up, the introduction of outer boar, seed selection and utilize work to obtain rapid progress, (1980 is 62.1% to improving national slaughter rate of hogs, 2003 is 129.9%), carcass weight (1980 for 57.1kg, 2003 be 76.6kg), carcass lean meat percentage, breeding coverage rate and economic benefit played important function.Take a broad view of in the current domestic pig industry scale lean meat species boar breeding of method; ternary hybrid commodity pork pig has comparative advantage in the ocean; the national boar genetic evaluation work of beginning in 1998 and the promulgation and the enforcement of " national boar genetic evaluation scheme (trying) "; the beginning of relatively large swine artificial insemination (AI) center set up; Landrace; Large White and carrying out of two flesh stern pig breeding cooperative groups activities etc.; make the performance test of boar outside China and genetic evaluation work just towards standardization and associating; unified direction stable development, the foundation of hereditary connection also begins to step a gratifying step between the group.Yet, clearly, we can say that China's pig industry has powerful pork pig production system, just " China is the big country of raising pigs " often said, still top core boar resource long-term dependence on import in " pyramid " breeding of method.In very long a period of time, for boar, China all is in the vicious circle of " introducing a fine variety → keep → degenerate → introduce a fine variety again ", and after accession to WTO, this situation is more serious.In a word, it is very unfavorable to the sustainable development of China's pig industry that significantly boar is introduced dependence, considers that from the long-term interest of country this kind phenomenon should not develop down again.In the face of this situation, force us must become dependence on import into creatively carrying out seed selection, seek the characteristic boar strain and the breeding of method that are fit to China's national situation, occupy a tiny space in international boar market.
Swine disease is the formidable enemy of Swine Production.In the breeding practice, pursue high yield merely and cause the resistance of pig to reduce usually, some original diseases are not eradicated, and have occurred new disease again, have seriously restricted the development of pig industry.Disease can cause that animal products is inferior, output descends, its also serious genetic progress that reduces simultaneously, and the direct economic loss that causes to pig industry accounts for 12%～15% of the gross output value, and therefore, carrying out prevention from suffering from the diseases is the prerequisite of development pig industry.The conventional method of disease preventing and treating is not that all diseases are all had effect and can not fundamentally control fully and the generation of eliminate disease and popular as improving environment, pharmacotherapy and immunity inoculation etc.Discover that the pathogenesis of many swine diseases is relevant with the genetic background of pig itself, promptly may there be resistance wholly or in part in pig to some diseases.In a broad sense, the generation of any disease all is the coefficient result of nature-nurture factor, and nearly all disease is all relevant with heredity.Say narrowly, hereditary disease be since in reproductive cell or the fertilized egg genetic material taken place due to the variation on structure or the function.Therefore, in the long run/term, adopt genetic method to improve pig the general resistance of disease is had the effect of effecting a permanent cure from hereditary basis.And general resistance is to keep the necessary condition that pig survives and produces in poor environment, and therefore cultivating the pig kind with general resistance becomes one of the major subjects in breeding field.But up to now, also not to the appraisement system of the general premunition of pig, can't carry out comprehensive assessment to the immune response ability of individuality, this has become pig and has improved bottleneck problem in the breeding practice of general premunition, foundes pig immunocompetence index, in-vitro method and has become with the system researches such as relation of actual premunition and cultivate the task of top priority and the key point with general resistance pig kind.
In the breeding practice that improves the general premunition of livestock and poultry, can utilize the indirect selection index (Rothschild, 1989) of immune response in theory as premunition.Biozzi etc. (1980) utilize studies confirm that of mouse antibody be medium heredity to the genetic mechanism of sheep red blood cell (SRBC) reaction, and select the humoral immunoresponse(HI) of certain antigen can improve the humoral immunoresponse(HI) of other antigens.The genetic mechanism of immune response has obtained extensive studies (Lamout, 1990) in poultry.At present the genetic mechanism of sheep red blood cell (SRBC) immune response has been carried out comprehensive research.In pig, between kind and intravarietal significance difference dissident obtained confirming with the immune response test of sheep red blood cell (SRBC) and DNP-hapten as antigen, but almost there is not or do not have non additivity (hybrid vigour) genetic control (Meek etc., 1984) of immune response.The additive genetic effect of the many immune characters of pig also all has report, as antibody response, and monocytic hyperplasia and poisonous effect, retardance surpasses quick reaction and total white blood cells and classification.The heritability of high by the time (0.3～0.8) promptly had bigger hereditary effect during a lot of immune characters had.It obviously is feasible improving the total resistance of communicable disease by the breeding method that improves antibody and cell immune response, but up to the present also do not set up pig immune response ability assessment system, realize that by improving pig immune response ability breeding for disease resistance also is in the exploratory stage.
Large White is the premium muscle type pig kind that China mainly introduces, and has critical role in market pig produces.The good characteristic of introducing the pig kind mainly is fast growth, lean meat percentage height, but also there is degradation problem and shortage under the premunition, the present invention is on the basis of the sick colony of Large White Chinese People's Anti-Japanese Military and Political College enterobacteria, measure immune response indexs such as cell factor such as different development stage interferon, interleukin and main antibody horizontal, analyze the immune response index and with the basis of the relation of growing on, determine the evaluation index of immune response and general premunition and include conventional seed selection and breeding system in that the effectively general premunition molecular labeling of screening also carries out the breeding efficiency assessment.Adopt the candidate gene method strong individuality of general premunition of selecting and remain to set up supporting system, be expected to reduce the incidence of disease of respiratory diseases such as asthma, cultivation has that the sick and general premunition of Chinese People's Anti-Japanese Military and Political College enterobacteria is strong, fast growth, lean meat percentage height, be suitable for the specialized supporting system of Large White that raises under the relative extensive condition, fundamentally lay the foundation, obtain considerable social benefit, economic benefit and social benefit for realizing that the pig efficient and healthful is cultured.
Summary of the invention
The objective of the invention is to set up the appraisement system of a kind of immune response and general premunition, the breeding method of the supporting system of Large White resistance that the science in the Large White of being provided at is feasible, supporting is the advantage that pig has anti-stress, strong, the anti-diarrhea of weaned piglets of accommodative ability of environment and oedema, lean meat percentage height, meat is excellent, the price of deed is high, litter size is many, and this method can also provide guidance for the breeding for disease resistance of domestic other pig kinds.
The breeding method of the supporting system of the high-quality and efficient stress-resistant of the said Large White of the present invention, be to constructional base group on the basis of the anti-F18 colibacillosis of core group major gene resistance FUT1 M307 genotype identification and premunition checking, immunology principle, molecular breeding technology and conventional breeding means are combined, utilize candidate gene strategy, cultivate the supporting system of the high-quality and efficient stress-resistant of Large White.
The present invention includes and give step: the first step: emphasis is taken all factors into consideration proterties such as birth weight, weaning weight and growth rate with candidate gene strategy, and establishment has the Large White underlying group of resistivity to diarrhea of weaned piglets and oedema.Second step: determine different preferendum granulocyte/lymphocyte (Heterophil/Lymphocyte, H/L) value, haemoglobin (HGB) and white blood cell count(WBC) (WTBC), IL6, IL12, IFN-γ, IFN-α are as the immune response and the general evaluation index of premunition, set up pig immune response appraisement system and determine the immune response index, include it in conventional seed selection and breeding system.The 3rd step: on the basis that obtains reproductive performance (ESR), lean meat percentage (ob) (being the genetic marker that has been applied at present) and disease-resistant trait related gene beneficial gene type (having locked BPI gene the 10th exon AA genotype at present), and then be identified for the seed selection group of favourable multiple gene polymerization.Thereby set up specialized supporting system at proterties such as general premunition, reproductive performance and lean meat percentages.Wherein specialized paternal underlying group is selected from disease-resistant proterties and lean meat percentage emphatically, and maternal underlying group is selected from disease-resistant performance and reproductive performance emphatically.The 4th step: the molecular marker breeding efficiency evaluation, determine the molecular association selection index, set up the MAS system, realize the polymerization of multiple beneficial gene, for the novel supporting system that cultivates high-quality provides technical method.The 5th step: adopt traditional breeding method and binding molecule marker assisted selection to continue determination and analysis and the raising of seed selection from generation to generation, supporting is the advantage that pig has anti-stress, strong, the anti-diarrhea of weaned piglets of accommodative ability of environment and oedema, lean meat percentage height, meat is excellent, the price of deed is high, litter size is many.
Said constructional base group and immunology principle, molecular breeding technology and conventional breeding means are combined among the present invention specifically may further comprise the steps:
(1) tissue DNA of extraction sample to be tested is standby;
(2) pcr amplification: the DNA with step (1) is a template, carries out pcr amplification with following primer,
Upstream primer is: 5 '-CCAACGCCTCCGATTCCTGT-3 ' (SEQ ID NO:1), downstream primer is: 5 '-GTGCATGGCAGGCTGGATGA-3 ' (SEQ ID NO:2);
(3) pcr amplification product restriction enzyme
The Hin6 IEnzyme is cut after polyacrylamide gel electrophoresis silver dyes analysis, select and remain and the individuality of 161bp band only occurs, in conjunction with the standard of conventional seed selection, take all factors into consideration proterties such as birth weight, weaning weight and growth rate, establishment has the underlying group of resistivity to diarrhea of weaned piglets and oedema;
(4) underlying group individuality in the picked at random step (3) utilizes the test of sticking of piglet intestinal epithelial cell that F18 Escherichia coli resistance is further verified;
(5) underlying group of step (3) is carried out the mensuration of immune response and general premunition index;
(6) DNA with step (1) is a template, carries out pcr amplification with following primer,
Upstream primer is: 5 '-CCCAACATGGAGATGCAGTTC-3 ' (SEQ ID NO:3), downstream primer is: 5 '-CAATGAATCAATGAGCACACC-3 ' (SEQ ID NO:4);
(7) pcr amplification product restriction enzyme
The Hap IIEnzyme is cut after polyacrylamide gel electrophoresis silver dyes analysis, and the sample that 445 bp bands only occur is that the core group of seed selection is selected and the molecule seed selection in early days;
(8) to the assessment of the core group in the step (7) molecular marker breeding efficient, set up the MAS breeding system, realize the polymerization of multiple beneficial gene, for the novel supporting system that cultivates high-quality provides technical method;
(9) utilize the technical method of step (8), the core group in the step (7) is continued determination and analysis and seed selection from generation to generation improves, constantly the uniformity of genetic stability and main proterties progressively realizes set breeding objective.Each selects and remain specialized paternal sow colony more than 50 from generation to generation, 3 above familys; The maternal sow of specialization colony is more than 100,5 above familys; Carry out paternal with maternal combining ability test and analysis simultaneously, optimization forms a complete set of; Through 4-5 generation, progressively realize set breeding objective.
Breeding objective be supporting be that disease incidence such as respiratory tract reduces more than 6%, multiparity is farrowed more than 11, feedstuff-meat ratio 2.9:1; Meat is good, intramuscular fat 〉=2.5%, the nothing yellowish pink is pale, quality is soft, tangent plane oozes out (PSE) and dark, strong, dry meat (DFD), be waterpower (drip loss)≤3%, marble grain scoring 2.8-3.0, yellowish pink scoring 3.0-4.0, the acid-base value (PH45) of butchering mensuration in back 45 minutes is between 6.3-6.6, butcher the acid-base value (PH) measured in back 24 hours between 5.4-5.8, tender degree≤3.6%.
Advantage of the present invention:
(1) starts with from pig resistance and carry out breeding for disease resistance, improve the immune response ability of pig, can more effectively solve the problem of a lot of diseases of pig such as respiratory disease and environmental interaction, fundamentally reduce the incidence of disease of many diseases.
(2) the present invention combines immunology principle, molecular breeding technology and conventional breeding means, utilizes candidate gene strategy, improves special premunition and the general premunition of Large White, and technical support and guidance can be provided for the breeding for disease resistance of other kinds.
Description of drawings
Fig. 1 is breeding underlying group FUT gene M 307 testing results for Large White resistance is supporting
M.?pBR322?DNA/BsuRI(HaeⅢ)Marker。
Fig. 2 is the 3 kinds of genotype Escherichia coli F18 resistances/neurological susceptibility checking result of underlying group FUT gene M 307 for Large White resistance is supporting.
Fig. 3 is 3 kinds of genotype detection results of underlying group BPI gene the 10th exon for Large White resistance is supporting
Wherein AA. 2,6,8,11,12, and BB. 4,10, and AB. 1,3,5,7,10; M is pUC19 DNA/
MspI (
The Hap II) marker.
Embodiment
1. constructional base group
Utilize molecular breeding technology and conventional breeding means, the genetic marker that utilization has been applied at present, at boar halothane (RYR1 gene) and the gene (FUT1 gene) relevant with diarrhea of weaned piglets and oedema are measured, after the halothane individuality is carried in rejecting, further according to FUT1 genetic test result, select FUT1 gene A A allelomorph (resistance type) individuality to match (Fig. 1), and by external sticking settling down test (being challenge test) in test and the body offspring is carried out the checking (Fig. 2) of anti-F18 coli-infection, the enantiopathy individuality continues selection and fixes then.Emphasis is taken all factors into consideration proterties such as birth weight, weaning weight and growth rate with candidate gene strategy, and establishment has the Large White underlying group of resistivity to diarrhea of weaned piglets and oedema.
The individual screening of FUT1 gene A A allelomorph (resistance type) is: with sample to be tested DNA is template, carries out pcr amplification with following primer,
Upstream primer is: 5 '-CCAACGCCTCCGATTCCTGT-3 ' (SEQ ID NO:1), downstream primer is: 5 '-GTGCATGGCAGGCTGGATGA-3 ' (SEQ ID NO:2);
The pcr amplification product restriction enzyme
The Hin6 IEnzyme is cut after polyacrylamide gel electrophoresis silver dyes analysis, and selecting and remain the individuality of 161bp band only occurs.
2. determine the evaluation index of immune response and general premunition
Measure cell factor and main antibody horizontals such as blood routine index, interferon, interleukin such as the different development stage periphery is blood red, total white blood cells.Inherent immunity responsibility individual in the underlying group is carried out overall merit in advance, analyze the immune response index and with grow and the basis of property relationship such as fertility on, determine different preferendum granulocyte/lymphocyte (Heterophil/Lymphocyte, H/L) value, haemoglobin (HGB) and white blood cell count(WBC) (WTBC), IL6, IL12, IFN-γ, IFN-α are as the immune response and the general evaluation index of premunition, set up pig immune response appraisement system, include it in conventional seed selection and breeding system.
3. general premunition proterties, reproductive performance and lean meat percentage molecular marker gene type detect
The reproductive performance (ESR), lean meat percentage (ob) gene (being the genetic marker that has been applied at present) that diarrhea of weaned piglets and oedema are had the Large White underlying group of resistivity detect, the relation of analyze and research individual ESR gene, ob gene pleiomorphism genotype and reproductive performance (litter size, produce the young number of work etc.) and lean meat percentage (thickness of backfat); Adopt analysis technology such as PCR-SSCP and PCR-RFLP simultaneously, general premunition proterties candidate gene such as related genes such as TLR4, BPI, SLA-DQA, SLA-DRA, SLA-DQB, SLA-DRB and TAP are detected, the polymorphism of analysis and research gene, identify idiotype and analyze the corresponding gene type and the relation of general premunition index, finally select the most significantly BPI gene the 10th exon of hereditary effect for use
HpaII AA genotype is as the genetic marker of general premunition.Obtaining on the basis of reproductive performance (ESR), lean meat percentage (ob) and disease-resistant trait related gene beneficial gene type, and then be identified for the seed selection group of favourable multiple gene polymerization.
BPI gene the 10th exon
HpaII AA genotype genetic marker screening technique is: be template with the sample to be tested, upstream primer is: 5 '-CCCAACATGGAGATGCAGTTC-3 ' (SEQ ID NO:3), downstream primer is: 5 '-CAATGAATCAATGAGCACACC-3 ' (SEQ ID NO:4); Carry out pcr amplification; The pcr amplification product restriction enzyme
The Hap IIEnzyme is cut after polyacrylamide gel electrophoresis silver dyes analysis, and the sample that 445 bp bands only occur is BPI gene the 10th exon
HpaII AA genotype.
4. general premunition proterties, reproductive performance and lean meat percentage and the polymerization of beneficial gene molecular labeling:
By the detection to the BPI gene, the individuality (♀ and ♂) that continues to select and remain and have disease-resistant gene type AA carries out the cultivation of disease-resistance population;
Simultaneously female hormone receptor gene (ESR) is selected in early days in piglet birth back (to the male and female pig), the efficiency of selection that improves litter size of pig and produce the young number of living reduces boar and cultivates cost; Aspect the meat selection,, directly carry out the selection of boar live body lean meat percentage to reduce slaughter determining to the ob genetic test.Thereby set up specialized supporting system at proterties such as general premunition, reproductive performance and lean meat percentages.Wherein specialized paternal underlying group is selected from disease-resistant proterties and lean meat percentage emphatically, and maternal underlying group is selected from disease-resistant performance and reproductive performance emphatically.
5. molecular marker breeding efficiency evaluation:
Utilize each genotype value of least-square analysis and make effect mutually between them, when making effect mutually when remarkable, A uses a model; When making effect mutually significantly the time, B uses a model.Model A:Y=μ+individual effects+genotype I effect+genotype II effect++ effect+residual error between effect+kind made mutually between different genotype; Model B: effect+residual error between Y=μ+individual effects+merging genotype effect+kind
Utilize the theoretical character value of each genotype correspondence and the phenotypic correlation of purpose character value of calculating of latus rectum, according to the latus rectum model correlation coefficient is carried out subdivision, drawing each genotype to the direct effect of purpose proterties with by the indirect effect of other genotype to the purpose proterties, and then each genotype of comparative analysis is to the decision degree of purpose proterties.
Latus rectum model: r
Xn.
X1P
y.
X1+ r
Xn.
X2P
y.
X2+ ... ..+P
y.
Xn=r
Xn.
y
6. the foundation of MAS breeding system:
Utilize favourable molecular genetic marker and conventional breeding value to estimate, determine the molecular association selection index (I=MS+EBV, wherein on behalf of molecule, MS mark, EBV is an estimated breeding value), set up the MAS system, realize the polymerization of multiple beneficial gene, for the novel supporting system that cultivates high-quality provides technical method.Adopt traditional breeding method and binding molecule marker assisted selection to continue determination and analysis and the raising of seed selection from generation to generation, constantly the uniformity of genetic stability and main proterties progressively realizes set breeding objective.Carry out paternal with maternal combining ability test and analysis simultaneously, optimization forms a complete set of.Supporting is the at present specialized paternal sow of core group colony more than 50,3 above familys; The maternal sow of specialization colony is more than 100,5 above familys; On Large White tradition merit basis (omnidistance feedstuff-meat ratio is below 3.0, and carcass lean meat percentage is more than 64%), behind the project smooth implementation, strive for reaching under the feeding standard level, supporting is that disease incidence such as respiratory tract reduces more than 6%, and multiparity is farrowed more than 11, feedstuff-meat ratio 2.9:1; Meat is good, intramuscular fat 〉=2.5%, the nothing yellowish pink is pale, quality is soft, tangent plane oozes out (PSE) and dark, strong, dry meat (DFD), be waterpower (drip loss)≤3%, marble grain scoring 2.8-3.0, yellowish pink scoring 3.0-4.0, the acid-base value (PH45) of butchering mensuration in back 45 minutes is between 6.3-6.6, butcher the acid-base value (PH) measured in back 24 hours between 5.4-5.8, tender degree≤3.6%.
SEQUENCE?LISTING
＜110〉Yangzhou University
＜120〉breeding method of the supporting system of the high-quality and efficient resistance of Large White
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gtgcatggca?ggctggatga 20
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cccaacatgg?agatgcagtt?c 21
<210> 4
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＜213〉artificial sequence
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Claims (2)
1. the breeding method of the supporting system of the high-quality and efficient stress-resistant of Large White, it is characterized in that constructional base group on the basis of the anti-F18 colibacillosis of core group major gene resistance FUT1 M307 genotype identification and premunition checking, immunology principle, molecular breeding technology and conventional breeding means are combined, utilize candidate gene strategy, cultivate the supporting system of the high-quality and efficient stress-resistant of Large White.
2. method according to claim 1 is characterized in that specifically may further comprise the steps:
(1) tissue DNA of extraction sample to be tested is standby;
(2) pcr amplification: the DNA with step (1) is a template, carries out pcr amplification with following primer,
Upstream primer is: 5 '-CCAACGCCTCCGATTCCTGT-3 ', downstream primer is: 5 '-GTGCATGGCAGGCTGGATGA-3 ';
(3) pcr amplification product restriction enzyme
The Hin6 IEnzyme is cut after polyacrylamide gel electrophoresis silver dyes analysis, select and remain and the individuality of 161bp band only occurs, in conjunction with the standard of conventional seed selection, take all factors into consideration proterties such as birth weight, weaning weight and growth rate, establishment has the underlying group of resistivity to diarrhea of weaned piglets and oedema;
(4) underlying group individuality in the picked at random step (3) utilizes the test of sticking of piglet intestinal epithelial cell that F18 Escherichia coli resistance is further verified;
(5) underlying group of step (3) is carried out the mensuration of immune response and general premunition index;
(6) DNA with step (1) is a template, carries out pcr amplification with following primer,
Upstream primer is: 5 '-CCCAACATGGAGATGCAGTTC-3 ', downstream primer is: 5 '-CAATGAATCAATGAGCACACC-3 ';
(7) pcr amplification product restriction enzyme
The Hap IIEnzyme is cut after polyacrylamide gel electrophoresis silver dyes analysis, and the sample that 445 bp bands only occur is that the core group of seed selection is selected and the molecule seed selection in early days;
(8) to the assessment of the core group in the step (7) molecular marker breeding efficient, set up the MAS breeding system, realize the polymerization of multiple beneficial gene, for the novel supporting system that cultivates high-quality provides technical method;
(9) utilize the technical method of step (8), the core group in the step (7) is continued determination and analysis and seed selection from generation to generation improves, constantly the uniformity of genetic stability and main proterties progressively realizes set breeding objective; Each selects and remain specialized paternal sow colony more than 50 from generation to generation, 3 above familys; The maternal sow of specialization colony is more than 100,5 above familys; Carry out paternal with maternal combining ability test and analysis simultaneously, optimization forms a complete set of; Through 4-5 generation, progressively realize set breeding objective.
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| CN102578041A (en) * | 2012-03-19 | 2012-07-18 | 扬州大学 | Method for using Huai pigs to cultivate new pig breed and produce hybrid commercial pigs |
| CN103757002A (en) * | 2013-12-26 | 2014-04-30 | 中国科学院昆明动物研究所 | Litter size related molecular marker of swine 6# chromosome |
| CN103757002B (en) * | 2013-12-26 | 2016-08-17 | 中国科学院昆明动物研究所 | A molecular marker relevant to litter size on No. 6 chromosomes of pig |
| CN103931560A (en) * | 2014-05-07 | 2014-07-23 | 扬州大学 | Sutai pig common disease-resistance combined selection index selecting breeding method |
| CN104186423A (en) * | 2014-09-18 | 2014-12-10 | 扬州大学 | Method for cultivating high-yield disease-resistant new-strain Meishan pigs |
| CN106417157A (en) * | 2016-08-02 | 2017-02-22 | 孙世铎 | Guanzhong black pig purification and rejuvenation method |
| CN106719444A (en) * | 2016-11-29 | 2017-05-31 | 广西壮族自治区农业科学院甘蔗研究所（中国农业科学院甘蔗研究中心） | Automatic collector of Corcyra cephalonica |
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