CN103911374B - The molecule marker relevant to Milk Fat Percentage in Dairy Cows proterties and application thereof - Google Patents
The molecule marker relevant to Milk Fat Percentage in Dairy Cows proterties and application thereof Download PDFInfo
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- CN103911374B CN103911374B CN201410116914.4A CN201410116914A CN103911374B CN 103911374 B CN103911374 B CN 103911374B CN 201410116914 A CN201410116914 A CN 201410116914A CN 103911374 B CN103911374 B CN 103911374B
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Abstract
The invention provides a kind of animal molecular marker, be specifically related to the clone of milk cow TRAPPC9 gene fragment and the application as molecule marker.Is the nucleotide sequence of described molecule marker as sequence table SEQ? ID? shown in No.1, there is 1 T204-G204 base mutation at the 204th, cause Milk Fat Percentage in Dairy Cows proterties to occur polymorphism.
Description
Technical field
The present invention relates to animal molecular marker field, specifically, relate to the application of a kind of TRAPPC9 gene relevant to Milk Fat Percentage in Dairy Cows proterties as molecule marker.
Background technology
Butterfat is a kind of natural high quality fat be present in milk, and compared to other fat, milk is more easily digested, and its digestibility can reach 98%.In milk-producing, the nutritive value of the higher milk of the content of butterfat in milk is also higher.At present, milk fat content weighs an important indicator of milk quality, and the height of its content is directly connected to the economic benefit of Cow product.
Along with the development of molecular genetics, Protocols in Molecular Biology and quantitative genetics, molecular genetic marker and marker assisted selection are widely applied in Animal Breeding, and achieve huge achievement.Snapshot sequencing technologies is a kind of typing method based on fluorescent mark single-basic extension principle, also little order-checking is claimed, mainly for the SNP somatotype project of moderate fluxes, its cardinal principle is mixed by certain system Sequenase, four kinds of fluorescently-labeled ddNTP, the extension primer being close to polymorphic site and PCR primer templates, namely primer extension base stops, detect through sequenator, the base kind Inner participating in reaction can be learnt according to the color at peak, thus judgement sample is in the genotype in this site.The method has that somatotype is accurate, flux is high, by advantages such as the restriction of SNP site polymorphism characteristic and number of samples restrictions.
Translocator particle composites 9 (TRAPPC9, traffickingproteinparticlecomplex9) gene is the gene that the recessive mental retardation dysnoesia of huamn autosomal is relevant, the protein transporters particle composites 9 of its coding participates in the vesica transfer of endoplasmic reticulum to golgi body and the differentiation of neurocyte, the albumen of its coding is also called NIBP(NIKandIKK β-bindingprotein), participate in vesicular traffic process, there is the function that enhance TNF-α activates NF-κ B signal path simultaneously.NF-κ B is important nuclear factor, participates in regulation and control lots of genes and expresses, play important biological function in cells survival, propagation, differentiation and apoptotic process.Also do not report about the impact of TRAPPC9 gene pleiomorphism on milk fat content at present.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of molecule marker relevant to Milk Fat Percentage in Dairy Cows proterties and application thereof.
In order to realize the object of the invention, first the present invention provides a kind of molecule marker relevant to Milk Fat Percentage in Dairy Cows proterties, its nucleotide sequence, as shown in sequence table SEQ IDNo.1, has 1 T204-G204 base mutation at the 204th, causes Milk Fat Percentage in Dairy Cows proterties to occur polymorphism.
Further, the site of described base mutation is positioned at the 57bp place of the 7th intron of TRAPPC9 gene.
Present invention also offers the primer pair for the described molecule marker that increases, its nucleotide sequence is as follows:
Forward primer F:5 '-TCTTCTCTTTTGACCCTACAG-3 ';
Reverse primer R:5 '-GGTTTCAAGTTCTGACTCCAG-3 '.Present invention also offers previous molecular and be marked at application in china holstein cows milk fat content marker assisted selection.
Further, described application comprises the steps:
1) genomic dna of milk cow to be detected is got;
2) take genomic dna as template, utilize described primer pair to carry out pcr amplification, obtain the object fragment of 274bp on china holstein cows TRAPPC9 gene;
3) utilize SnaPshot technology for detection PCR primer, if 204bp place is T in amplified production sequence, then milk cow to be measured belongs to the high china holstein cows Dominant variety of milk fat content.
Wherein, described step 2) in PCR reaction system be 25 μ L, wherein 100ng/ μ L genomic DNA template 1 μ L, 10pmol/ μ L primers F and each 1 μ L of R, 12.5 μ LPremixTaqTM, 9.5 μ LddH
2o.
Wherein, described step 2) in PCR reaction conditions be: 95 DEG C of denaturation 10min; 94 DEG C of sex change 30s, 60 DEG C of renaturation 30s, 72 DEG C extend 30s, 35 circulations; 72 DEG C extend 10min; Be cooled to 4 DEG C of maintenances.
Wherein, SnaPshot technology for detection PCR primer is utilized in described step 3), concrete steps are: get step 2) the PCR primer 3ul ExoI that obtains and FastAP purifying, mainly remove the residue primer in reaction product with ExoI, remaining DNTP in reaction is removed with FastAP, utilize SNP somatotype to extend primer after purifying to extend, after extension, upper machine checks order.If shown in sequencing result figure as left in Fig. 1, be TT type, then milk cow to be measured belongs to the china holstein cows advantage individuality of high milk fat content, occurs that i.e. TG or GG type is then average individual as shown in figure and right figure in Fig. 1.
The present invention, by carrying out gene type to the SNP site of china holstein cows TRAPPC9 gene, utilizes the GLM process of SAS9.1 software that the milk fat content of genotypic results and milk cow is carried out association analysis and finds that the milk fat content pole of TT type individuality is significantly higher than GG type individuality (P<0.05).Being found to be of this site is carried out molecular breeding and is provided new mark.
Further, the nucleotides sequence of described SNP somatotype extension primer is classified as:
5’-TTTTTTTTTTTTTTTTAGCAAAACCAGATGGACTTGTG-3’。
The present invention also provides the test kit detecting china holstein cows high milk fat content Dominant variety, described test kit comprises forward primer F, reverse primer R, SNP somatotype extends in primer and PremixTaqTM, ExoI, FastAP, ExoIbuffer, SnapshotMix one or more;
Wherein PremixTaqTM is by dNTPs, archaeal dna polymerase, Mg
2+, PCR damping fluid composition.
Beneficial effect of the present invention is:
SNP marker site provided by the present invention, finds first in china holstein cows cows, and has pole remarkably influenced to milk fat content, provides new material and scientific basis for carrying out china holstein cows milk fat content marker assisted selection.
SNP marker provided by the invention by the restriction such as age, sex, not can be used for early stage seed selection, accelerates breeding process; Detection method is accurately and reliably, convenient and easy.
Accompanying drawing explanation
Fig. 1 is china holstein cows three kinds of genotype SnaPshot sequencing and typing figure in the present invention; Wherein, left figure is TT type, and middle figure is TG type, and right figure is GG type (genotype of usual peak figure is distinguished by color, and its Green is TT type, and black is GG type).
Fig. 2 is three kinds of genotype sequencer maps; Be respectively TT type, TG type and GG type.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If without specified otherwise, the technique means in following embodiment is conventional means in this area, and material therefor is all buied from conventional reagent company.
The acquisition of the SNP marker that embodiment 1 is relevant to china holstein cows milk fat content proterties and qualification
The extraction of 1.1 china holstein cows poba gene group DNA to be measured
Tail venous collection china holstein cows blood to be measured, normal temperature is placed and is treated blood coagulation in 3-4 hour, and now blood is divided into serum and sludged blood two portions, serum is clear yellow, sludged blood is garnet, only has in white corpuscle and contains DNA, be present in sludged blood in bovine blood.
Utilize sky root blood/cell/tissue genome DNA sample to extract test kit and extract genomic dna from sludged blood, concrete steps are as follows:
Put into sterilized 2mL round bottom centrifuge tube with the sludged blood of eye scissors clip 0.2-0.3mL, add the cell pyrolysis liquid CL of 500 μ L;
Utilize hand-held Syrup-homogenizing instrument by abundant for clot homogenate, the centrifugal 1min of vibration 15s, 12000rpm, discards upper strata garnet supernatant liquor;
Again add 700 μ L cell pyrolysis liquid CL, fully vibration makes lower sediment scatter suspension, the centrifugal 1min of 12000rpm, abandoning supernatant;
Add 200 μ L damping fluid GS, abundant vortex fully suspends to precipitation;
Add 20 μ L Proteinase Ks and 250 μ L damping fluid GB, fully mix;
Sealed by centrifuge tube sealed membrane to put in 56 DEG C of hybrid heaters and digest 3-4 hour, for guaranteeing abundant digestion, need in digestive process to put upside down mixing for several times, final solution becomes limpid transparent, brief centrifugation;
Add 200 μ L and ice dehydrated alcohol, turn upside down mixing 15s, now may occur flocks;
Proceeded in adsorption column CB3 by previous step gained solution, adsorption column puts into collection tube, and the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube, adsorption column is put into collection tube;
In adsorption column CB3, add 500 μ L Deproteinization damping fluid GD, leave standstill the centrifugal 30s of 2min, 12000rpm, discard the waste liquid in collection tube, adsorption column is put into collection tube;
In adsorption column CB3, add 700 μ L rinsing liquid PW, the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube, adsorption column is put into collection tube;
In adsorption column CB3, add 500 μ L rinsing liquid PW, the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube;
Adsorption column is put into collection tube, and the centrifugal 2min of 12000rpm, discards waste liquid, adsorption column is placed in room temperature and places several minutes, thoroughly remaining on volatilization adsorption column ethanol;
Proceeded to by adsorption column in a new centrifuge tube, add the TE damping fluid of 100 μ L56 DEG C preheatings, room temperature places the centrifugal 2min of 5min, 12000rpm, and genomic dna is then present in centrifuge tube.
The amplification of 1.2 object fragments
According to the ox TRAPPC9 gene order that NCBI provides, oligo6 is utilized to design primer, comprise forward primer F:5 '-TCTTCTCTTTTGACCCTACAG-3 ' and reverse primer R:5 '-GGTTTCAAGTTCTGACTCCAG-3 ', with genomic dna in 1.1 for template carries out pcr amplification, object fragment sequence as shown in SEQIDNo.1, SNP site is positioned at 204bp place, and base is T or G herein.
PCR reaction system is 25 μ L, wherein 100ng/ μ L genomic DNA template 1 μ L, 10pmol/ μ L primers F and each 1 μ L of R, 12.5 μ LPremixTaq
tM, 9.5 μ LddH2O.
PCR reaction conditions is: 95 DEG C of 10min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations; 72 DEG C of 10min; 4 DEG C of maintenances.
1.3 gene type
Utilize SnaPshot technology to carry out gene type, concrete steps are as follows:
PCR primer purifying: get the PCR primer ExoI and FastAP purifying that obtain in 1.2, mainly removes the residue primer in reaction product with ExoI, removes remaining DNTP in reaction with FastAP.
Reaction system is PCR primer 3ul, ExoI0.2ul, FastAP0.8ul, ExoIbuffer0.7ul, and moisturizing is to 7ul.
Reaction conditions is 37 DEG C of 15min, 80 DEG C of 15min.
Extension: system is purified pcr product 2ul, SnapshotMix reagent 1ul, extend primer mixing 2ul, moisturizing is to 6ul.
Condition is 96 DEG C of 1min; 96 DEG C of 10sec, 52 DEG C of 5sec, 60 DEG C of 30sec, 30 circulations;
Get 1ul extension products, add 10ul loading loading, 95 DEG C of sex change 3min, immediately ice-water baths, upper sequenator.
According to Snapshot sequencing result interpretation genotype, as shown in Figure 1, be reverse primer owing to extending primer, therefore the left figure of sequencing result is TT type individuality, middle figure is that GG type is individual, and right figure is that TG type is individual.
For verifying the accuracy of somatotype, get sample segment after somatotype and carry out pcr amplification, amplified production is delivered to order-checking company and check order, sequencing result as shown in Figure 2: be respectively TT type, TG type and GG type, sequencing result is consistent with genotyping result, illustrates that genotyping result accurately and reliably.
The association analysis of embodiment 2SNP site and china holstein cows milk fat content
Carry out SNP somatotype to 332 china holstein cowses of the different dairy cow farm in Northern Part of China in the present embodiment, TRAPPC9 gene 204bp place genotyping result of sequence as shown in SEQIDNO.1 is as shown in table 1.
The GLM process of utilization SAS9.1 software carries out the association analysis between genotype and milk fat content, and model is as follows:
y
ijl=μ+g
i+h
j+p
l+e
ijl，
Wherein, y
ijlfor the phenotypic number of milk production trait; μ is population mean; g
ifor genotype effects; h
jfor season in field year effect; p
lfor parity effect; e
ijlfor random residual.
The gene frequency of table 1SNP genotype in institute's Research Group and gene frequency
As shown in Table 1, the homozygous number of individuals of CC is significantly higher than TT type, C allelotrope is protogene, the side's of card comptibility test χ 2=2.06< χ 20.05 (1)=3.84, expected value and observed value difference are not remarkable, illustrate that this SNP site is in Hardy-Weinberg equilibrium state in colony.
Table 2SNP genotype and the association analysis of china holstein cows milk fat content
Note: different lowercase alphabet shows significant difference, i.e. P<0.05.
As shown in Table 2, the impact of genotype on milk fat content reaches pole conspicuous level, and TT type milk fat content is significantly higher than GG type (P<0.05).
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (6)
1. a molecule marker application in china holstein cows milk fat content marker assisted selection relevant to Milk Fat Percentage in Dairy Cows proterties; The nucleotide sequence of described molecule marker, as shown in sequence table SEQ IDNo.1, has 1 T204-G204 base mutation at the 204th, causes Milk Fat Percentage in Dairy Cows proterties to occur polymorphism; The site of described base mutation is positioned at the 57bp place of the 7th intron of TRAPPC9 gene.
2. application according to claim 1, is characterized in that, comprises the steps:
1) genomic dna of milk cow to be detected is got;
2) take genomic dna as template, utilize primer pair to carry out pcr amplification, obtain the object fragment of 274bp on china holstein cows TRAPPC9 gene;
The nucleotide sequence of described primer pair is as follows:
Forward primer F:5 '-TCTTCTCTTTTGACCCTACAG-3 ';
Reverse primer R:5 '-GGTTTCAAGTTCTGACTCCAG-3 ';
3) utilize SnaPshot technology for detection PCR primer, if 204bp place is T in amplified production sequence, then milk cow to be measured belongs to the high china holstein cows Dominant variety of milk fat content.
3. application according to claim 2, is characterized in that, described step 2) in PCR reaction system be 25 μ L, wherein 100ng/ μ L genomic DNA template 1 μ L, 10pmol/ μ L primers F and each 1 μ L of R, 12.5 μ LPremixTaqTM, 9.5 μ LddH
2o.
4. application according to claim 2, is characterized in that, described step 2) in PCR reaction conditions be: 95 DEG C of denaturation 10min; 94 DEG C of sex change 30s, 60 DEG C of renaturation 30s, 72 DEG C extend 30s, 35 circulations; 72 DEG C extend 10min; Be cooled to 4 DEG C of maintenances.
5. application according to claim 2, it is characterized in that, described step 3) in utilize SnaPshot technology for detection PCR primer, concrete steps are: get step 2) the PCR primer 3 μ l ExoI that obtains and FastAP purifying, utilize SNP somatotype to extend primer after purifying to extend, after extension, upper machine checks order.
6. application according to claim 5, is characterized in that, the nucleotides sequence that described SNP somatotype extends primer is classified as: 5 '-TTTTTTTTTTTTTTTTAGCAAAACCAGATGGACTTGTG-3 '.
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| CN104232776A (en) * | 2014-09-18 | 2014-12-24 | 中国农业大学 | Molecular marker related to milk production traits of dairy cows and application of molecular marker |
| CN104561304A (en) * | 2014-12-31 | 2015-04-29 | 中国农业大学 | Molecular marker for detecting DNA methylation of cow mastitis resistance |
| CN104611425B (en) * | 2015-01-15 | 2017-08-08 | 中国农业大学 | A kind of SNP marker related to china holstein cowses S. aureus L-forms Mastitis resistance and its application |
| CN104928289A (en) * | 2015-06-04 | 2015-09-23 | 内蒙古河套农牧业技术研究院 | Method for detecting sheep FecB gene polymorphism by use of SNaPshot technology |
| CN106754909B (en) * | 2017-01-24 | 2019-06-21 | 青岛市畜牧兽医研究所 | One kind molecular labeling relevant to the forthright shape of milch goat butterfat and its application |
| CN107254525B (en) * | 2017-06-22 | 2020-08-25 | 陕西师范大学 | Method for evaluating milk quality based on cattle BAF60c gene locus mutation |
| CN112175963A (en) * | 2020-09-30 | 2021-01-05 | 安徽省天长市周氏羊业有限公司 | SPP1 gene and method for evaluating developmental quality of golgi in single follicle of sheep |
| CN112899373B (en) * | 2021-01-28 | 2023-08-15 | 武汉轻工大学 | SNP (Single nucleotide polymorphism) marker related to milk fat percentage of southern Holstein cows in China and application thereof |
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Non-Patent Citations (4)
| Title |
|---|
| AC_000171.1;NCBI;《GenBank》;20100604;全文 * |
| RefSNP（rs#）rs133738259;NCBI;《dbSNP》;20101109;全文 * |
| 中国荷斯坦奶牛κ-酪蛋白基因第四外显子多态性与产奶性状的关联分析;鞠志花等;《遗传》;20081231;摘要，第1314页1.2部分 * |
| 中国荷斯坦牛leptin基因多态性及其与产奶性能的关系;徐凯勇;《中国优秀硕士学位论文全文数据库 农业科技辑》;20080115(第1期);全文 * |
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