CN104293963B - A kind of method of 145 SNP judgment of Wuzhishan minipig inbred line of application - Google Patents
A kind of method of 145 SNP judgment of Wuzhishan minipig inbred line of application Download PDFInfo
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Abstract
The method that the invention discloses 145 SNP judgment of Wuzhishan minipig inbred line of a kind of application.Auxiliary provided by the invention identifies that whether pig to be measured is the method for Inbreding Wuzhishan pig, comprises the steps: the genotype detecting pig to be measured based on 145 SNP site；If meeting all standards of (1) to (145), pig to be measured is the Inbreding Wuzhishan pig of candidate；If being unsatisfactory for all standards of above-mentioned (1) to (145), pig to be measured is the non-Inbreding Wuzhishan pig of candidate.The present invention has substantial worth for the germplasm identification of Inbreding Wuzhishan pig.
Description
Technical field
The method that the present invention relates to 145 SNP judgment of Wuzhishan minipig inbred line of a kind of application.
Background technology
During 1989-2003 years, " Wuzhi Mountain inbred cultivation, Specific character and exploitation " in the Ministry of Agriculture's " the Seventh Five-Year Plan, eight or five, 95 " industry brainstorm project, and the Department of Science and Technology " 863 ", National Nature fund committee emphasis, under the major project subsidy about 7 problems such as grade, research work makes a breakthrough, that is: with 1 male 1 female Wuzhi Mountain pig (also known as WZSP) for being ancestral, continue with " son joins mother ", the aggregate measures such as " full sib " copulation, progressively overcome inbreeding to breed and cause that reproductive survival rate is extremely low, less than 20% harmful effect, set up F17Inbred line colony and pedigree, and carried out utilizing the technological means of the multiple advanced persons such as molecular genetic means DNA fingerprint figure similarity coefficient, micro-satellite to monitor this inbred line kind F7-F16Inbred cultivation research process, the preliminary genetic development disclosing Inbreding Wuzhishan pig and feature, demonstrate the verity of this inbred cultivation process, science.And carried out substantial amounts of exploitation research, it is shown that as the type that is unsurpassed in excellence of ideals of human being Research of Animal Model for Study, obtain certain economic benefit and bigger social benefit.Its research contents is identified by Ministry of Agriculture's technological achievement respectively at nineteen ninety-five and 2005 " WZSP Specific character and exploitation research " (agriculture section fruit identifies [95] No. 075), " WZSP experiment inbred cultivation is studied with Molecular and genetic basis " two achievements, wherein within 1999, obtains Ministry of Agriculture's scientific and technological progress third prize.
Miniature pig inbreeding is cultivated and is tentatively researched and developed achievement, at home and abroad creates large effect, and e.g., the U.S., Italy, Japan, West Germany, Korea S etc. require to introduce a fine variety or joint study one after another, and especially the U.S. proposes 5,000,000 dollars of 50 boars bought at that time for 1996；Korea S disagrees to introduce a fine variety latter 2000 to be proposed to provide funds 5,000,000 yuan to do factory etc. Chinese joint.It main reason is that inbred line animal is special Animal Genetic Resource, it can be the same with precision instrument physically to chemical analysis pure reagent, can draw rapidly for life science, sensitive, science data accurately, there is significantly high use value and Research Significance, so having cultivated more than 450 kind of inbred line system mice, rat etc. in the world at present.Small-sized mammalian inbred line, as animal model, is widely used to solve mankind's difficult and complicated illness, capture life sciences and the research field such as medical science, a pharmacy difficult problem.Due to the difference in many ways such as physiology between planting, inbred pig is cultivated very difficult, new confirms that (2013.11) have no reported success so far in the world through looking into.But pig is compared with muroid has bigger similarity with the mankind, solving mankind's difficult and complicated illness, capturing life sciences and the research such as medical science, a pharmacy difficult problem will play replaceable, not unique effect.Inbred pig is larger animal gene genetic resource rare, valuable in the world.Before this, far away from the many units of the sixties in last century some developed countries of USA and Europe, just go out huge fund and carried out inbred pig nurturing research, but fail, the coefficient of inbreeding is the highest is only capable of reaching 0.75.And China was by 15 years nurturing researchs, the miniature pig resource utilizing China special has obtained F17, the coefficient of inbreeding reached 0.965, more the good news is and overcome, cross over the stage occurred frequently that the caused piglet of close breeding is dead.Therefore, inbred pig is cultivated and is successfully probably realized first in China.Through further work in recent years, obtain now Inbreding Wuzhishan pig F17To F22。
Owing to not yet there is cultivation reported success the inbred pig world, more building without inbred line is standard and authentication method.Therefore, must use for reference, follow strictly inbred mouse, the breeding method of rat carries out, namely breed 20 generations more than with 2 pig close relatives of ancestral all one's life and set up complete pedigree, detect its hereditary stability, set up authentication method, set up strain standard especially and to distinguish between existing Hainan Mt. Wu-zhi Shan pig kind, science, credible evidence could be provided to common people, just can complete the unprecedented rear temporary genetic resources Innovation project without the person that has come, promote the well-being of mankind.
Once the success of miniature pig inbred cultivation, China will have independent intellectual property right, have the pig genetic germplasm resource of the innovation of the leading level in the world, will fill up world research in this research field blank, abundant larger animal inbred cultivation theory and practice；" for difficulty person " as ideals of human being, it is the important foundation stone that makes progress of biotechnology research and basis for innovation, cooperative development and the innovation of the aspects such as China's life sciences, physianthropy, pharmacy will be greatly promoted, promote the well-being of mankind make irreplaceable status and effect for solution mankind's difficult and complicated illness, extending life.Therefore, have important practical significance and theory significance.
Summary of the invention
The method that it is an object of the invention to provide 145 SNP judgment of Wuzhishan minipig inbred line of a kind of application.
The invention provides a kind of auxiliary and identify that whether pig to be measured is the method for Inbreding Wuzhishan pig, comprise the steps:
Detect the pig to be measured genotype based on 145 SNP site；
If meeting all standards of following (1) to (145), pig to be measured is the Inbreding Wuzhishan pig of candidate:
If being unsatisfactory for all standards of above-mentioned (1) to (145), pig to be measured is the non-Inbreding Wuzhishan pig of candidate.
Described pig to be measured can be Inbreding Wuzhishan pig or Hainan Mt. Wu-zhi Shan pig.
Described Inbreding Wuzhishan pig can belong to F20To F22In arbitrary generation.
Described Inbreding Wuzhishan pig can belong to F20Generation or F20Generation later from generation to generation.
In described method, certain chip can be adopted to detect the pig to be measured genotype based on 145 SNP site；Described certain chip is be fixed with the nucleic acid chip of the single strand dna shown in sequence 1 to the sequence 145 of sequence table respectively in difference.
The present invention also protects a kind of nucleic acid chip, is fixed with the single strand dna shown in sequence 1 to the sequence 145 of sequence table respectively in difference.
The present invention also protects the application of described nucleic acid chip, for as follows (a) or (b): (a) auxiliary identifies whether pig to be measured is Inbreding Wuzhishan pig；B () auxiliary identifies whether swinery body to be measured is Inbreding Wuzhishan pig colony.
Described pig to be measured can be Inbreding Wuzhishan pig or Hainan Mt. Wu-zhi Shan pig.
Described Inbreding Wuzhishan pig can belong to F20To F22In arbitrary generation.
Described Inbreding Wuzhishan pig can belong to F20Generation or F20Generation later from generation to generation.
Present invention also offers a kind of auxiliary and identify that whether swinery body to be measured is the method for Inbreding Wuzhishan pig colony, comprise the steps:
From swinery body to be measured, stochastic sampling obtains sample to be tested, then each sample to be tested genotype based on 145 SNP site is detected, if all samples to be tested are satisfied by all standards of (1) to (145) as follows, swinery body to be measured is the Inbreding Wuzhishan pig colony of candidate:
If there are the more than one all standards being unsatisfactory for above-mentioned (1) to (145) in sample to be tested, swinery body to be measured is the non-Inbreding Wuzhishan pig colony of candidate.
In described method, from swinery body to be measured, stochastic sampling obtains statistically significant sample to be tested.
In described method, from swinery body to be measured, stochastic sampling obtains more than 16 samples to be tested.
Described pig to be measured can be Inbreding Wuzhishan pig or Hainan Mt. Wu-zhi Shan pig.
Described Inbreding Wuzhishan pig can belong to F20To F22In arbitrary generation.
Described Inbreding Wuzhishan pig can belong to F20Generation or F20Generation later from generation to generation.
In described method, certain chip can be adopted to detect the pig to be measured genotype based on 145 SNP site；Described certain chip is be fixed with the nucleic acid chip of the single strand dna shown in sequence 1 to the sequence 145 of sequence table respectively in difference.
The present invention has substantial worth for the germplasm identification of Inbreding Wuzhishan pig.
Detailed description of the invention
Below example is easy to be more fully understood that the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
Inbreding Wuzhishan pig:
Li Kai, Feng Shutang, Mu Yulian, Yang Shulin, Han Jianlin, Liu Lan, the new rising sun of member, Guo Yong, 3 inbreeding family microsatellite allele heredity changes of WZSP, Scientia Agricultura Sinica, 2012,42 (5): 1751-1760.
Hainan Mt. Wu-zhi Shan pig:
Hou Guan, Wang Dongjing, Guan Song, glorious, Huang Xianzhou, Wuzhi Mountain pig small group hereditism detects, ecology of domestic animals, the 2007, the 28th volume the 6th phase: 44-47.
The laboratory animal of Inbreding Wuzhishan pig is 48, wherein F20Generation 16, F21Generation 16, F22Generation 16.The laboratory animal of Hainan Mt. Wu-zhi Shan pig is 16.
Embodiment 1, method foundation
, there are 145 SNP site, according to its genotype, it is possible to authenticate Inbreding Wuzhishan pig and non-Inbreding Wuzhishan pig in the present inventor's discovery when Inbreding Wuzhishan pig and non-Inbreding Wuzhishan pig carry out genome research.The genotype of 145 SNP site of Inbreding Wuzhishan pig is homozygous (genotype described in table 1).145 SNP site of non-Inbreding Wuzhishan pig are heterozygous.
In 145 SNP site, 12 are positioned at No. 1 chromosomal 1st section, 16 are positioned at No. 1 chromosomal 15th section, 14 and are positioned at No. 1 chromosomal 50th section, 16 are positioned at No. 1 chromosomal 78th section, and 15 are positioned at No. 2 chromosomal 2nd section, and 14 are positioned at No. 2 chromosomal 12nd section, 13 are positioned at No. 2 chromosomal 19th section, 20 are positioned at No. 18 chromosomal 5th section, and 13 are positioned at No. 18 chromosomal 14th section, and 12 are positioned at No. 18 chromosomal 20th section.
145 probes (probe is adjacent with SNP site with the calmodulin binding domain CaM of genomic DNA, is positioned at upstream or the downstream of SNP site, is therefore extended by end, it is possible to knows the genotype of SNP site) are devised based on 145 SNP site the present inventor.
The source sequence residing on genomic DNA of the title of 145 SNP, residing chromosome, position on chromosome, the genotype of Inbreding Wuzhishan pig, the Source Type of SNP, the nucleotide sequence of 145 probes and SNP is in Table 1.
Table 1
In table 1, N represents A, T, C or G.In table 1, the underscore in source sequence marks region corresponding to probe (containing two kinds of forms, probe is identical with the nucleotide sequence of underlined region, the nucleotide sequence reverse complemental of probe and underlined region).In table 1, when the Source Type of SNP is for [T/G], heterozygous is TG, and when the Source Type of SNP is for [A/G], heterozygous is AG, and when the Source Type of SNP is for [T/C], heterozygous is TC, and when the Source Type of SNP is for [A/C], heterozygous is AC.
Embodiment 2,145 SNP judgment of Wuzhishan minipig inbred line of application and non-Inbreding Wuzhishan pig
1, taking laboratory animal, ear vein takes blood, extracts genomic DNA from blood sample.
2, take the genomic DNA that step 1 obtains, hybridize with the nucleic acid chip being fixed with 145 probes (single strand dna shown in the sequence 145 of sequence 1 to the sequence table of 145 probe respectively sequence tables).
3, after completing step 2, each point in nucleic acid chip is carried out end extension, thus knowing the genotype of 145 SNP site in genomic DNA.
4, take the genomic DNA that step 1 obtains, carry out genome sequencing, know the genotype of 145 SNP site in genomic DNA.
It is shown that the result of the result of step 3 and step 4 is completely the same.
The genotype of 145 SNP site of 48 laboratory animals of Inbreding Wuzhishan pig is homozygous (genotype described in table 1 of embodiment 1).145 SNP site of 16 laboratory animals of Hainan Mt. Wu-zhi Shan pig are heterozygous.
Claims (9)
1. auxiliary identifies that whether pig to be measured is a method for Inbreding Wuzhishan pig, comprises the steps:
Detect the pig to be measured genotype based on 145 SNP site；
If meeting all standards of following (1) to (145), pig to be measured is the Inbreding Wuzhishan pig of candidate:
If being unsatisfactory for all standards of above-mentioned (1) to (145), pig to be measured is the non-Inbreding Wuzhishan pig of candidate.
2. the method for claim 1, it is characterised in that: described Inbreding Wuzhishan pig belongs to F20To F22In arbitrary generation.
3. the method for claim 1, it is characterised in that: described Inbreding Wuzhishan pig belongs to F20Generation or F20Generation later from generation to generation.
4. the method as described in arbitrary in claims 1 to 3, it is characterised in that: in described method, adopt certain chip to detect the pig to be measured genotype based on 145 SNP site；Described certain chip is be fixed with the nucleic acid chip of the single strand dna shown in sequence 1 to the sequence 145 of sequence table respectively in difference.
5. a nucleic acid chip, is fixed with the single strand dna shown in sequence 1 to the sequence 145 of sequence table respectively in difference.
6. the application of nucleic acid chip described in claim 5, for as follows (a) or (b):
A () auxiliary identifies whether pig to be measured is Inbreding Wuzhishan pig；
B () auxiliary identifies whether swinery body to be measured is Inbreding Wuzhishan pig colony.
7. auxiliary identifies that whether swinery body to be measured is a method for Inbreding Wuzhishan pig colony, comprises the steps:
From swinery body to be measured, stochastic sampling obtains sample to be tested, then each sample to be tested genotype based on 145 SNP site is detected, if all samples to be tested are satisfied by all standards of (1) to (145) as follows, swinery body to be measured is the Inbreding Wuzhishan pig colony of candidate:
If there are the more than one all standards being unsatisfactory for above-mentioned (1) to (145) in sample to be tested, swinery body to be measured is the non-Inbreding Wuzhishan pig colony of candidate.
8. method as claimed in claim 7, it is characterised in that: described pig to be measured is Inbreding Wuzhishan pig or Hainan Mt. Wu-zhi Shan pig.
9. method as claimed in claim 7 or 8, it is characterised in that: in described method, adopt certain chip to detect each sample to be tested genotype based on 145 SNP site；Described certain chip is be fixed with the nucleic acid chip of the single strand dna shown in sequence 1 to the sequence 145 of sequence table respectively in difference.
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| WO2016061711A1 (en) * | 2014-10-21 | 2016-04-28 | 北京盖兰德生物科技有限公司 | Method for identifying wuzhishan miniature pig inbred line by using 145 snp |
| CN105255877B (en) * | 2015-11-16 | 2018-02-13 | 上海市农业科学院 | It is used for SNP marker and its application traced to the source on No. 6 chromosomes of pig |
| CN105255878B (en) * | 2015-11-16 | 2018-01-26 | 上海市农业科学院 | It is used for SNP marker and its application traced to the source on No. 6 chromosomes of pig |
| CN108130375A (en) * | 2017-12-04 | 2018-06-08 | 漳州傲农现代农业开发有限公司 | A kind of method for differentiating Jinhua Pigs, Landrace based on SNP site |
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| CN101532055A (en) * | 2009-02-04 | 2009-09-16 | 中国农业科学院北京畜牧兽医研究所 | Method for implementing assistant identification on inbred lines of Wuzhishan miniature pigs and special kit thereof |
| CN101608237A (en) * | 2009-02-04 | 2009-12-23 | 中国农业科学院北京畜牧兽医研究所 | A kind of method of assisting judgment of Wuzhishan minipig inbred line |
| CN103898107A (en) * | 2014-04-17 | 2014-07-02 | 江西农业大学 | Major SNP (single nucleotide polymorphism) marker influencing growth traits of pigs and application thereof in genetic improvement of productivity of breeding pigs |
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| CN101532055A (en) * | 2009-02-04 | 2009-09-16 | 中国农业科学院北京畜牧兽医研究所 | Method for implementing assistant identification on inbred lines of Wuzhishan miniature pigs and special kit thereof |
| CN101608237A (en) * | 2009-02-04 | 2009-12-23 | 中国农业科学院北京畜牧兽医研究所 | A kind of method of assisting judgment of Wuzhishan minipig inbred line |
| CN103898107A (en) * | 2014-04-17 | 2014-07-02 | 江西农业大学 | Major SNP (single nucleotide polymorphism) marker influencing growth traits of pigs and application thereof in genetic improvement of productivity of breeding pigs |
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