CN101255478A - Detection method of porcine white clothing hairs homozygous genotype - Google Patents
Detection method of porcine white clothing hairs homozygous genotype Download PDFInfo
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Abstract
The invention discloses a detection method for pig white clothing hair homozygosis, comprising the steps: acquiring pig ear tissue genome DNA, determining the difference between different white clothing hair genotypes, PCR amplification of 18th introne of pig white clothing hair gene KIT, and detecting the homozygosis I/I of pigs with white clothing hair. The invention is helpful to determine the true white gene heredity information of the pigs with white clothing hair, provides technical support for controlling the clothing hair color of offspring, and fixing the advantages of white clothing hair pigs, and has important productive significance.
Description
Technical field
The invention belongs to the biology field of pig.Specifically, the present invention relates to pig white by the homozygosity detection method of hair gene type.
Background technology
The hair color type of pig can be divided into two classes substantially: whole white and non-whole white, non-whole white have comprised shades of colours such as wild boar look, all black, whole red, white background band blackspot and black piebald.
Hair color is all by Gene Handling, wherein the apparent recessive relation of each gene is white seat gene＞non-white seat gene (Spillman et al.1906), Wright proposed the hypothesis of the white hair color of pig by two dominant genes controls in 1918, Hetzer is at Yorkshire in 1945 and landrace) usually the I gene be (I/I) that isozygotys.Pig, black greatly pig, duroc, Pietrain pigs and colored Chinese variety are the homozygotes (i/i) of recessive gene in colored species such as Berkshire, the ripple.Find in the I site except I and i allelotrope, to also have I at present
P(Johansson et al.1992) waits other allelotrope, it should be noted that I allelotrope is dominance for other allelotrope.
KIT gene and I allele.The KIT gene of pig is made up of 21 exons, the long 2919bp of entire coded sequence.The fragment of 2892bp in the encoding sequence has been carried out having at least three allelotrope I, I on clone and this site of RT-PCR product order-checking discovery
P, i.Their dominance grade is: I＞I
P＞i.Allelotrope I is corresponding to the white hair color of complete dominance, on the molecular structure except that having normal KIT gene (being called KIT1), also carry the total length KIT gene copy (being called KIT2) that contains two kinds of sudden changes, one of sudden change occurs on the introne 18, lack four bases, cause KIT genetic expression imbalance, be called and regulate sudden change.Another sudden change occurs on first nucleotide position of introne 17, causes the montage sudden change of G → A, ignores exons 17 during montage, causes tyrosine kinase activity impaired, is called structural mutation.I
PAllelotrope shows as patch or spot proterties white or coloured mao, and patch and spot boundary are obvious, the no-buffer band.Existing normal KIT gene (KIT1) on the molecular structure, also have a duplicate KIT gene (KIT1) total length copy, this copy has increased KIT expression of gene amount, thereby influence the utilizability of the part of loose stem cell factor, upset the migration and the survival of melanocyte precursor, produce patch or spot phenotype.I allelotrope only carries a normal KIT gene (KIT1), and the melanocyte precursor can normally move and survive, and shows normal hair color.
Because white hair color pig has the ability that absorbs the sunlight middle-ultraviolet lamp strong, rickets ability height, morbidity easily changes from skin to be found out, the mammary gland non-pigment, trunk is attractive in appearance, on the world market welcome and pig white hair color more coloured by live pig butcher the lower advantages such as (Rothchild et al.1998) of cost, domestic raiser is more prone to raise white pig kind.In recent years, some raisers also blackspot can occur from pure white pig kind breeding back offspring external introduction and existing, this is because this pure white pig is the non-homozygous individual of allelotrope I, therefore because allelotrope I has dominance action for other allelotrope, its individual hair color always shows as pure white, covered should individuality the breeding true background, have only as this individuality and other allelotrope I non-its offspring of homozygous individual post-coitum just can in the hair color phenotype, show blackspot.At present, also there is not one at this problem and the objective conveniently solution of energy.
Summary of the invention
The invention provides the detection method of boar white dorsal body setae homozygous genotype.
The objective of the invention is to be achieved through the following technical solutions: a kind of detection method of porcine white clothing hairs homozygous genotype, it is characterized in that, may further comprise the steps:
(1) obtains pig ear tissue genomic dna;
(2) determine that different whites are by the difference of hair gene type;
(3) pig white is by the pcr amplification of hair gene KIT the 18th intron;
(4) white is detected by the homozygosity of live pig genotype I/I.
Further, described step (3) is specially: design a pair of primer according to pig KIT gene the 18th intron sequences among the state-run biotechnology GenBank of information center of the U.S. and come this sudden change regional sequence of amplification in vitro, primer sequence is as follows: K18F:5 '-TGTGGGAGCTCTTCTCTTTAGG-3 '; K18R:5 '-ACTGGCATTCCGGGGTAG-3 ' is that template is carried out pcr amplification with this primer with the pig genomic dna.
Further, described step (4) is specially: whole white is dyeed through ethidium bromide EB by the pcr amplification product that obtains in the live pig genomic dna, at 2.5% agarose 10V/cm horizontal strip electrophoresis after 1.5 hours, can clearly see and estimate two products of size for 161bp and 165bp, utilize system to carry the optical density(OD) intensity of each individual two product of analysis software after gel imaging system is taken pictures and calculate its ratio, when 165bp reaches 0.9～1 scope than the ratio of 161bp, can determine that this individuality is the white genotype I/I individuality that isozygotys, if this ratio less than 0.9, can determine that then this individuality is the non-white genotype of isozygotying.
The invention has the beneficial effects as follows: porcine white clothing hairs homozygous genotype method for quick of the present invention can be used for different from the detection of hair color pig, and controls its offspring by the hair color.The present invention helps clear and definite white by the real white gene genetic information of live pig, and for controlling the offspring by the hair color, fixed white is provided technical support by the advantage that live pig had, and has important production meaning.
Description of drawings
Fig. 1 is that pig white is formed synoptic diagram by the allelotrope of hair gene combination intron; Wherein, A, I/I genotype; B, I/I
PGenotype; C, the I/i genotype.
Fig. 2 be white with non-white by live pig PCR product agarose electrophoresis figure; Wherein, M, pBR322 DNA/BsuRIMarker; 1, blackspot Pietrain pigs PCR product result; 2-5, white is by live pig PCR product result.
Fig. 3 is that genotype I/I homozygosity detects synoptic diagram; Wherein, A, Different Individual PCR product agarose electrophoresis detects; B～C analyzes two intron amplified production optical density(OD) intensity of the corresponding Different Individual of each swimming lane.
Embodiment
The detection method of pig white dorsal body setae homozygous genotype provided by the invention may further comprise the steps:
1. pig ear tissue genomic dna obtains.
2. determine that different whites are by the difference of hair gene type.
According to background technology as can be known, pig whole white clothing hairs homozygous genotype allelotrope is combined as I/I and is equivalent to 2 KIT1 and 2 KIT2, and 18 introns of two normal 18 introns and two disappearance 4 bases are promptly arranged, and its ratio should be 1; The pig whole white is had only an allelotrope I to be equivalent to 1 KIT1 and 1 KIT2 in the non-homozygous genotype of hair, therefore and another non-I allelotrope can not have KIT2 again, normal 18 introns of this individual KIT gene and have the ratio of 18 introns of 4 base deletions should be greater than 1 (Fig. 1).
3. pig white is by the pcr amplification of hair gene KIT the 18th intron.
(accession number: X93082) designed a pair of primer and come this sudden change regional sequence of amplification in vitro, primer sequence is as follows: K18F:5 '-TGTGGGAGCTCTTCTCTTTAGG-3 ' according to pig KIT gene the 18th intron sequences among the state-run biotechnology GenBank of information center of the U.S.; K18R:5 '-ACTGGCATTCCGGGGTAG-3 ', is that template is carried out pcr amplification with this primer with the pig genomic dna, estimate to obtain two products of 161bp and 165bp in by the live pig genomic dna at whole white, and estimate to have to the product of a 165bp in by the live pig genomic dna at non-whole white.In electrophoresis process with respect to complete in vain by two products of the individual PCR of hair, non-whole white is had only a 165bp band by the genomic PCR product of live pig, it is not subjected to the obstruction of other 161bp band and swimming faster, therefore on electrophorogram its present position with non-complete in vain by the 161bp product position of live pig PCR product suitable (Fig. 2).
4. white is detected by the homozygosity of live pig genotype I/I.
The PCR product that whole white is obtained in the live pig genomic dna dyes through ethidium bromide (EB), at 2.5% agarose 10V/cm horizontal strip electrophoresis after 1.5 hours, can clearly see and estimate two products of size for 161bp and 165bp, utilize system to carry the optical density(OD) intensity of each individual two product of analysis software after gel imaging system is taken pictures and calculate its ratio (Fig. 3), when 165bp reaches 0.9 ~ 1 scope than the ratio of 161bp, can determine that this individuality is the white genotype I/I individuality that isozygotys, if this ratio can determine that less than 0.9 this individuality is the non-white genotype of isozygotying.Because the selectivity of PCR competition, in the pcr amplification of white clothing hairs homozygous genotype individuality, because the normal 18 intron copy numbers of long segment are identical with short segmental 4 base deletions, 18 intron copy numbers, primer can be partial to slightly in conjunction with short segmental 4 base deletions, 18 introns, thereby the 165bp product will occur approaching 1 rate value greater than 0.9 than 161bp product ratio.And in the pcr amplification of the non-homozygous genotype individuality of white quilt hair, because the normal 18 intron copy numbers of long segment are the several times of short segmental 4 base deletions, 18 introns, carry out the polymerase chain reaction so primer can preferentially be attached on 4 base deletions, 18 intron sequences, so the 165bp product will be less than 0.9 than 161bp product ratio.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the invention.
Zhejiang University experimentation on animals pasture picked at random white by 12 of hair Yorkshire offsprings and contrast purebred white by 2 of hair Yorkshires and black piebald by 2 of hair Pietrain pigs, adopt pig ear tissue sample 0.3 gram, extract genomic dna in the ear tissue with traditional phenol/chloroform extraction method, gained genomic dna purity is diluted to final concentration 50ng/ μ L after the TE dissolving about about 80%～97%.
With according to pig KIT gene the 18th intron sequences among the state-run biotechnology GenBank of information center of the U.S. (accession number: X93082) She Ji primer (K18F:5 '-TGTGGGAGCTCTTCTCTTTAGG-3 '; K18R:5 '-ACTGGCATTCCGGGGTAG-3 '), be that template is carried out pcr amplification with the pig genomic dna, the pcr amplification reaction system is 15 μ L, reaction system consists of the 50ng genomic dna, 1 * PCR buffer, 1.5mmol/L MgCl
2, 0.2pmol/L dNTP and 1U Taq enzyme, each 0.2 μ mol/L of forward and reverse primer.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min, carry out program then and be: 94 ℃ of sex change 40s, 55 ℃ of renaturation 40s, 72 ℃ are extended 40s, 30 circulations, last 72 ℃ are extended 7min, 4 ℃ of preservations.
The homozygosity of embodiment 3 genotype I/I detects
Each individual PCR product dyes through ethidium bromide (EB), at 2.5% agarose 10V/cm horizontal strip electrophoresis after 1.5 hours, can clearly see that white is all had two bands of estimating big or small 161bp of being and 165bp by live pig, black piebald Pietrain pigs has only a band product (Fig. 2).Utilize system to carry the optical density(OD) intensity of each individual two band of analysis software after gel imaging system is taken pictures and calculate normal intron product than sudden change intron product ratio (Fig. 3), should be reached 0.9 ~ 1 scope by the homozygous 18 intron PCR product 165bp of hair gene type I/I than the ratio of 161bp according to white, obtain having white by individual 4 of hair gene type I/I homozygote (result is suitable with the contrast Yorkshire), all the other are whole white by the non-homozygote individuality of the genotype of hair, shown in the homozygosity detected result signal table of table 1 genotype I/I.
Table 1
The whole white that filters out with this method is by non-homozygote of hair gene I individual 71 and the mating of black piebald Pietrain, the hair color separation phenomenon has appearred in its offspring, and offspring's hair color phenotype white piglet and non-white piglet quantitative proportion are about 1: 1, meet mendelian inheritance, confirm that further individual 71 is that white is by the non-homozygote of hair gene I; The whole white that filters out equally is by individual 4-18 of the genotype I/I homozygote of hair and 57 mating, its offspring is that whole white is by the hair individuality entirely, after measuring, these individual employing the inventive method all obtain ratio greater than 0.9, shown in offspring's genotype I/I homozygosity detected result signal table of individual 4-18 of table 2 gene I homozygote and 57 mating, be that these individualities are white by the genotype I/I homozygote individuality of hair, we also use the individual and black piebald Pietrain mating of this 4-18, its offspring is whole white by the hair individuality, and these results confirm further that 4-18 is that white is by the gene I homozygote individuality of hair really.Like this, we have just obtained having white by the homozygous individuality of genotype I/I of hair, and they and any color be by the pig mating of hair, and its offspring is whole white.This has just controlled the offspring by the color of hair, fixed white by the hair individuality have rickets ability height, morbidity be easy to attractive in appearance from Skin observing, mammary gland non-pigment, trunk, butcher that cost is low, the raiser more be inclined to raise and the world market on advantage such as welcome.
Table 2
At last, note also that what more than enumerate only is a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1. the detection method of a porcine white clothing hairs homozygous genotype is characterized in that, may further comprise the steps:
(1) obtains pig ear tissue genomic dna.
(2) determine that different whites are by the difference of hair gene type.
(3) pig white is by the pcr amplification of hair gene KIT the 18th intron.
(4) white is detected by the homozygosity of live pig genotype I/I.
2. the detection method of porcine white clothing hairs homozygous genotype according to claim 1, it is characterized in that, described step (3) is specially: design a pair of primer according to pig KIT gene the 18th intron sequences among the state-run biotechnology GenBank of information center of the U.S. and come this sudden change regional sequence of amplification in vitro, primer sequence is as follows: K18F:5 '-TGTGGGAGCTCTTCTCTTTAGG-3 '; K18R:5 '-ACTGGCATTCCGGGGTAG-3 ' is that template is carried out pcr amplification with this primer with the pig genomic dna.
3. the detection method of porcine white clothing hairs homozygous genotype according to claim 1, it is characterized in that, described step (4) is specially: whole white is dyeed through ethidium bromide EB by the pcr amplification product that obtains in the live pig genomic dna, at 2.5% agarose 10V/cm horizontal strip electrophoresis after 1.5 hours, can clearly see and estimate two products of size for 161bp and 165bp, utilize system to carry the optical density(OD) intensity of each individual two product of analysis software after gel imaging system is taken pictures and calculate its ratio, when 165bp reaches 0.9～1 scope than the ratio of 161bp, can determine that this individuality is the white genotype I/I individuality that isozygotys, if this ratio less than 0.9, can determine that then this individuality is the non-white genotype of isozygotying.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191070A (en) * | 2016-07-26 | 2016-12-07 | 中国人民解放军第三军医大学 | Cause pig KIT mutant gene and the application thereof of deafness |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191070A (en) * | 2016-07-26 | 2016-12-07 | 中国人民解放军第三军医大学 | Cause pig KIT mutant gene and the application thereof of deafness |
| CN106191070B (en) * | 2016-07-26 | 2019-09-10 | 中国人民解放军第三军医大学 | Cause deaf pig KIT mutated gene and its application |
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