CN100400674C - Molecular marker of Wuzhishan pig and its application - Google Patents
Molecular marker of Wuzhishan pig and its application Download PDFInfo
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Abstract
The present invention discloses a molecular marker of a Wuzhishan small-sized pig and the application thereof, which aims at providing the molecular marker and the method for identifying the Wuzhishan small-sized pig by the molecular marker. The Wuzhishan small-sized pig molecular marker provided by the present invention has the nucleotide sequence of sequence 1 in a sequence table. The method for identifying the Wuzhishan small-sized pig by the molecular marker uses genome DNA of the pig to be checked as a form panel, and uses a pair of primer to carry out PCR amplification to check whether an 88 bp band exists in amplification products, and the primer is composed of nucleotide sequence which has sequence 2 in the sequence table and the nucleotide sequence which has sequence 3 in the sequence table. The present invention establishes foundation for better exploration and utilization of Wuzhishan small-sized pig and Wuzhishan small-sized inbred line.
Description
Technical field
The present invention relates to a kind of molecule marker and the application thereof of WZSP, particularly utilize the method for this molecular markers for identification WZSP.
Background technology
WZSP (WZSP) is distinctive famous rare, the in imminent danger pig kind of China, and this kind originates in the five fingers mountain area by north, middle part, Hainan Province.Only deposit 10 remainders to the eighties, endangered, after go through hardships, protect in the Institute of Animal Husbandry, China Academy of Agriculture Scinces strange land and plant successfully, and successfully cultivated into inbred lines.Because pig and people have bigger similarity on features such as dissect physiology, Nutrition and Metabolism, biochemical indicator, especially the cardiovascular systems structure more similar to the people (Peter J.M et al.Xenotransplantation.[J] British Medical Bulletin, 1999,55 (2): 446-459, and WZSP has small, inheritance stability, early sexual maturity, features such as strong stress resistance, become the desirable object that the animal disease model of ideals of human being and efficacy of new drug, production of vaccine detect, the desirable organ donor source of especially human xenotransplant.At present, WZSP be listed in embargo kind and national two classes lay special stress on protecting one of animal (Feng shu-tang.Chinese Wuzhishan Pig, 1998. Feng's book halls. Chinese Wuzhi Mountain pig, 1998).
The current method of utilizing little satellite to do the population evaluation mainly contains minimum distance method and maximum likelihood method, with these two kinds of methods, can calculate the probability that certain individuality belongs to a certain population.
Et al. carries out individual cluster research to the Norway horse, and its result shows when little satellite seat during at 20, use maximum likelihood method, accuracy rate can reach more than 98% (
Et al.Gunby E﹠amp; Red K.H.Genetic structure of Norwegian horsebreeds.Journal of Animal Breeding and Genetics.2000,117,307-317.Find when the Yang Shu forest rent is studied 18 place of china pig variety genetic diversities with 27 microsatellite DNA marks: when choosing maximum likelihood method, the accuracy rate of differentiation is 98.45%-99.91%; And when choosing the genetic distance method, the accuracy rate of differentiation be 96.82%-99.30% (Yang Shulin. 18 place of china pig variety analysis of genetic diversity: [doctorate paper]. Hua Zhong Agriculture University, 2004).With above two kinds of methods, can carry out the population evaluation to species well, but all there is the shortcoming of aspects such as required microsatellite DNA mark is many, experimental procedure is loaded down with trivial details in they.
Summary of the invention
A kind of molecule marker that the purpose of this invention is to provide WZSP.
The molecule marker of WZSP provided by the present invention, name is called Sw936-88, has the nucleotide sequence of sequence 1 in the sequence table.
Sequence 1 in the sequence table is by 88 based compositions.
Can utilize by the nucleotide sequence with sequence 2 in the sequence table and have a pair of primer that the nucleotide sequence of sequence 3 in the sequence table is formed, be the molecule marker that template obtains this 88bp with the genomic dna of WZSP.
Sequence 2 in the sequence table is by 21 based compositions, and the sequence 3 in the sequence table is by 20 based compositions.
Second purpose of the present invention provides the method for a kind of WZSP of evaluation.
The method of evaluation WZSP provided by the present invention, be that genomic dna with pig to be measured is a template, the a pair of primer that utilization is made up of nucleotide sequence with sequence 2 in the sequence table and the nucleotide sequence with sequence 3 in the sequence table carries out pcr amplification, detects the band whether a 88bp is arranged in the amplified production.
Described 88bp band has the nucleotide sequence of sequence 1 in the sequence table.
If the band of this 88bp is arranged in the amplified production, pig then to be measured is WZSP.
Described amplified production can detect by 15% non-denaturing polyacrylamide gel.
People such as Jiang Shenghua utilize 18 microsatellite DNA marks, to studying of WZSP (22), fragrant pig (28), people pig (12), Large White (32), landrace (31), Du Luoke (32), Korea S people pig (31), Korea S Large White 218 colonies of 8 kinds such as (30), find little satellite seat S0070 264, SW2 103,85 only in WZSP, the occurring of SW936.The present invention is on this basis, is material with WZSP inbred lines and Tongcheng pig further, utilizes little satellite seat S0070, SW2, Sw936 to detect, and the result obtains the new little satellite specific alleles of a WZSP inbred lines Sw936-88.This WZSP inbred lines be Institute of Animal Husbandry, China Academy of Agriculture Scinces by two boar (a public mother) by the mode of height inbreeding cultivate the 17th generation inbred lines.Mutation frequency scope (1.2 * 10 according to little satellite seat
-4-1.5 * 10
-2), through inbred WZSP inbred lines of 17 generations, its probability of undergoing mutation can be ignored basically.Therefore, think that the allelotrope of WZSP inbred lines on little satellite seat Sw936 is that their original ancestry (a public mother) has, and pass on from generation to generation.110 WZSP inbred lines of experimental result proof have and carry molecule marker Sw936-88 in 70.9% individuality, and other 10 kinds (Tongcheng pig, duroc, landrace, Large White, greatly enhance logical pig, grow up and lead to pig, fragrant pig, people pig, Korea S people pig and Korea S Large White) in all do not carry molecule marker Sw936-88, illustrate that Sw936-88 is the important molecular markers that the WZSP population is identified.The present invention is for developing WZSP better and the WZSP inbred lines is laid a good foundation.
Description of drawings
Fig. 1 is the part electrophoresis result of WZSP inbred lines at little satellite seat Sw936
Fig. 2 is the part electrophoresis result of Tongcheng pig at little satellite seat Sw936
Fig. 3 is Sw936-88 cloning and sequencing result and comparison
Embodiment
Experimental technique among the following embodiment is ordinary method if no special instructions.
The acquisition of embodiment 1, molecule marker Sw936-88 of the present invention
Material
Research object WZSP inbred lines be Institute of Animal Husbandry, China Academy of Agriculture Scinces by two boar (a public mother) by the mode of height inbreeding cultivate the 17th generation inbred lines.The ear tissue sample of WZSP inbred lines (110) picks up from Institute of Animal Husbandry, China Academy of Agriculture Scinces Wuzhi Mountain pig and protects kind of a factory, and the blood sample of Tongcheng pig (100) picks up from Tongcheng County, Hubei Province boar factory of bureau of animal husbandry.
Primer
The primer of little satellite seat Sw936 of increasing is synthetic by Shanghai Bo Ya Bioisystech Co., Ltd, and its sequence is:
Forward primer: 5 ' TCTGGAGCTAGCATAAGTGCC 3 ' (sequence 2)
Reverse primer 5 ' GTGCAAGTACACATGCAGGG 3 ' (sequence 3)
According to document Sambrook J, Fritsch E F, Maniatis T.Molecular Cloning.ALaboratory Manual, 2nd ed.Cold Spring Harbor Laboratory Press, the method for New York.1989 is extracted the genomic dna of above-mentioned pig respectively.Genomic dna with extraction is a template, under the guiding of above-mentioned primer, carries out pcr amplification respectively, and reaction system is 20 μ l.Wherein template DNA is 30-50ng, 10 * damping fluid, 2 μ l, Mg
2+2 μ l (15mMol/L), dNTP 0.4 μ l (10mMol/L), each 5pM of upstream and downstream primer, Tag enzyme 1U.Wherein, 10 * damping fluid come from TaKaRa Taq test kit (TaKaRa company, CodeNo.DR100A).Reaction conditions is: 95 ℃ earlier, and 5min; 94 ℃ again, 30s, 55 ℃, 30s, 72 ℃, 30s, after 35 circulations, 72 ℃ are extended 5min.The product of amplification is got 4 μ l 140V voltage electrophoresis 14-16h on 15% non-denaturing polyacrylamide gel after 3% agarose gel electrophoresis detection, silver dyes colour developing.The scanning of Fotodyne gel imaging system is also preserved, and Gel Analyzer software analysis purpose clip size is to confirm WZSP inbred lines specific alleles.The comparative result of little satellite electropherotyping of WZSP inbred lines and Tongcheng pig shows that little satellite seat Sw936 has specific alleles in the WZSP inbred lines, and size is 88bp.The part electrophoresis result of little satellite seat Sw936 in two colonies as depicted in figs. 1 and 2.Among Fig. 1 and Fig. 2, M is dna molecular amount mark PBR322DNA/Hae III Markers.Native polyacrylamide gel electrophoresis by 15%, the scanning of Fotodyne gel imaging system, Gel Analyzer software analysis is in 110 WZSP inbred lines, have 77 individualities to contain the allelotrope of 88bp at little satellite seat Sw936, proportion is 70.9%.
Population genetic diversity POPGENE VERSION 1.31 and PPAP 3.0 software package analyses.In WZSP inbred lines (110), calculate the individuality shared ratio in colony contain specific alleles.The WZSP inbred lines is in allelotrope and the frequency of little satellite seat Sw936, with Tongcheng swinery body the fixation index of little satellite seat Sw936 (Fis, Fit, Fst) and average respectively shown in table 1-2:
Table 1WZSP inbred lines is in allelotrope and the gene frequency of little satellite seat Sw936
Table 2WZSP inbred lines, Tongcheng pig the fixation index of little satellite seat Sw936 (Fis, Fit, Fst)
| Little satellite seat | Sample number (head) | Fis | Fit | Fst |
| Sw936 | 420 | 0.1544 | 0.4093 | 0.3014 |
The result shows that little satellite seat Sw936 goes up the allelotrope of 88bp size because gene frequency (0.5) higher (table 1), with other allelotrope clip size gap big (Fig. 1), and when in the swinery body of Tongcheng, verifying, can be clear that very that its homologous segment size area do not find this equipotential gene (Fig. 2), thereby confirm that tentatively the allelotrope of this 88bp size is the specific alleles of WZSP inbred lines.Two colonies are 0.3014 (table 2) in the fixation index Fst of little satellite seat Sw936 average, than Taihu Lake Swine group (0.182) (Fan Bin. the research of microsatellite marker assessment Chinese Pigs genetic diversity and the some restraining factors of marker assisted selection efficient: [master thesis]. Hua Zhong Agriculture University, 1999,35) and Europe 11 kinds (0.27) (Laval G.N, et al.Genetic diversity of eleven european pig breeds.Genet.Sel.Evol.2000,32:187-203) all big, this illustrates that two colonies differentiation degree between the kind of little satellite seat Sw936 is very big.Exist the difference of bigger location distribution and modes of reproduction aspect closely related between this result and two colonies, WZSP originates in the five fingers mountain area, Hainan Province, protect kind in strange land, Beijing and successfully bred inbred lines, and Tongcheng pig originates in Tongcheng County, Hubei Province and random mating.
In order further to confirm WZSP inbred lines specific alleles, make the result have more preciseness.This specific alleles has been carried out cloning and sequencing.
Agarose Gel DNA Purification Kit Ver.2.0 (sepharose recovery test kit) with TaKara company reclaims amplified production from gel, after 4 ℃ in pGEM-T Easy carrier is connected 12 hours, transformed into escherichia coli (DH5 α), picking positive colony plasmid, bacterium colony PCR detects, bacterium liquid is served the order-checking of Hai Boya Bioisystech Co., Ltd, and the sequence at the corresponding little satellite of the pig among sequencing result and GenBank seat compares analysis.The result shows that little satellite seat Sw936 goes up 88bp allelotrope and lacks 11 repeating units (GT) than the allelotrope (110bp) of (GenBankAccession Number:AF225107) little satellite seat Sw936 among the GenBank (these 11 repeating units (GT) represent with underscore as shown in Figure 3 in Fig. 3.Among Fig. 3, A: little satellite seat Sw936 goes up 88bp allelotrope cloning and sequencing result; The sequence (110bp) of little satellite seat Sw936 among the B:GenBank).The sequence length scope of little satellite seat Sw936 that GenBank announces is 92-112bp, and this sequence is through 15% non-denaturing polyacrylamide detections, Gel Analyzer software analysis, cloning and sequencing turns out to be 88bp at last.Therefore, this specific alleles is the neomorph of little satellite seat Sw936.This explanation is not all found the allelotrope of this 88bp at little satellite seat Sw936 in the colony of having studied, this provides strong replenishing for affirmation of WZSP inbred lines specific alleles on little satellite seat Sw936.
The specificity of embodiment 2, molecule marker Sw936-88
Method according to embodiment 1 is extracted WZSP (100), duroc (50), Large White (50), landrace (50), greatly enhance logical pig (50), logical pig (50) grows up, fragrant pig (50), people pig (50), the genomic dna of Korea S people pig (50) and Korea S Large White (50) ear tissue sample, utilize primer: 5 ' TCTGGAGCTAGCATAAGTGCC 3 ' (sequence 2) and 5 ' GTGCAAGTACACATGCAGGG 3 ' (sequence 3) carry out pcr amplification, amplified production carries out 15% native polyacrylamide gel electrophoresis, the result shows in 100 WZSPs that detected, 71 specific bands that contain 88bp are arranged, and 50 durocs, 50 Large Whites, 50 landraces, 50 greatly enhance logical pig, the logical greatly pig of 50 head lengths, 50 fragrant pigs, 50 people pigs all do not have this band in 50 Korea S people pigs and 50 Korea S Large Whites.According to the method for embodiment 1, all 88bp specific bands that obtain are checked order, the result shows the nucleotide sequence that above-mentioned all 88bp bands all have sequence 1 in the sequence table, all the sequence with molecule marker Sw936-88 is consistent.
Sequence table
<160>3
<210>1
<211>88
<212>DNA
＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>1
tctggagcta gcataagtgc catcctctgg aaatacagaa gagcctgtgt gtgtgtgtgt 60
gtgtgtgtcc ctgcatgtgt acttgcac 88
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
tctggagcta gcataagtgc c 21
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
gtgcaagtac acatgcaggg 20
Claims (5)
1. a kind of molecule marker of WZSP, its nucleotide sequence is shown in sequence in the sequence table 1.
2. method of identifying WZSP, be that genomic dna with pig to be measured is a template, the a pair of primer that utilization is made up of the reverse primer shown in the sequence 3 in forward primer shown in the sequence in the sequence table 2 and the sequence table carries out pcr amplification, detects the band whether a 88bp is arranged in the amplified production.
3. method according to claim 2 is characterized in that: the nucleotide sequence of described 88bp band is shown in sequence in the sequence table 1.
4. according to claim 2 or 3 described methods, it is characterized in that: described amplified production detects by 15% non-denaturing polyacrylamide gel.
5. obtain the primer of the described molecule marker of claim 1, a pair of primer of forming by the nucleotide sequence of sequence 3 in the nucleotide sequence of sequence in the sequence table 2 and the sequence table.
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| CN103301507B (en) * | 2013-05-28 | 2015-04-29 | 北京博辉瑞进生物科技有限公司 | Artificial biological tendon and preparation method thereof |
| CN103272274B (en) * | 2013-05-28 | 2015-04-15 | 北京博辉瑞进生物科技有限公司 | Biological repair tablet for herniae and preparation method thereof |
| CN103272275B (en) * | 2013-05-28 | 2015-04-15 | 北京博辉瑞进生物科技有限公司 | Biological repair tablet for endocranium and preparation method thereof |
| CN103923993B (en) * | 2014-04-16 | 2016-03-23 | 北京盖兰德生物科技有限公司 | A kind of method applying the SNP judgment of Wuzhishan minipig inbred line of ABHD5 gene |
| CN103923994B (en) * | 2014-04-16 | 2016-04-20 | 北京盖兰德生物科技有限公司 | A kind of method applying the SNP judgment of Wuzhishan minipig inbred line of MLANA gene |
| CN111363830B (en) * | 2020-04-10 | 2022-06-21 | 上海市农业科学院 | Specific microsatellite primer composition for identifying experimental miniature pig breeds and method for identifying experimental miniature pig breeds |
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| CN1502231A (en) * | 2002-11-21 | 2004-06-09 | 中国农业科学院畜牧研究所 | Method for obtaining inbred line small size pig |
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| CN1502231A (en) * | 2002-11-21 | 2004-06-09 | 中国农业科学院畜牧研究所 | Method for obtaining inbred line small size pig |
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| 中国小型猪实验动物化培育及种质特异性研究. 冯书堂等.实验动物科学与管理，第20卷. 2003 * |
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